Hepatitis c virus variants

ABSTRACT

The present invention relates to HCV variants, particularly variants that are resistant to a protease inhibitors such as VX-950. Also provided are methods and compositions related to the HCV variants. Further provided are methods of isolating, identifying, and characterizing multiple viral variants from a patient.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. provisionalapplication No. 60/735,577, filed Nov. 11, 2005 and U.S. provisionalapplication No. 60/854,598, filed Oct. 25, 2006. The contents of theseprovisional applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to hepatitis C virus (HCV) variants.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) infects more than 170 million people worldwideand is the leading cause of chronic hepatitis, which can ultimately leadto end-stage liver cirrhosis and hepatocellular carcinoma. The standardtreatment for HCV infection is currently pegylated interferon alpha(Peg-IFN) in combination with ribavirin (RBV). The goal of HCV therapyis to eliminate viral infection by obtaining a sustained viral response(SVR) as defined by having undetectable HCV-RNA in the blood after 6months of antiviral treatment. Unfortunately, the current treatment isnot effective in about 50% of subjects with genotype 1, and the sideeffects are significant. Thus, new antiviral targets and improvedtreatment strategies are needed (Pawlotsky, J. M., and J. G.McHutchison, 2004, Hepatitis C. Development of new drugs and clinicaltrials: promises and pitfalls. Summary of an AASLD hepatitis singletopic conference, Chicago, Ill., Feb. 27-Mar. 1, 2003, Hepatology39:554-67; Strader, et al., 2004, Diagnosis, management, and treatmentof hepatitis C. Hepatology 39:1147-71).

The non-structural (NS) 3-4A protease is essential for HCV replicationand a promising target for new anti-HCV therapy. VX-950, a potent andspecific NS3-4A protease inhibitor demonstrated substantial antiviralactivity in a phase 1b trial of subjects infected with HCV genotype 1(Study VX04-950-101). The degree to which a subject responds totreatment and the rate at which viral rebound is observed could in partbe due to genotypic differences in sensitivity to the proteaseinhibitor. The rapid replication rate of HCV, along with the poorfidelity of its polymerase, gives rise to an accumulation of mutationsthroughout its genome (Simmonds, P., 2004, Genetic diversity andevolution of hepatitis C virus—15 years on. J. Gen. Virol. 85:3173-88).The degree to which sequence variability in the protease region affectsthe catalytic efficiency of the enzyme or the binding of an inhibitor isnot known. Additionally, the generation of numerous viral genomes withremarkable sequence variation presents potential problems of emergingdrug resistant virus in subjects treated with antiviral therapy. Indeed,drug resistance against antiviral drugs, such as HIV proteaseinhibitors, is well documented (Johnson, et al., 2004, Top. HIV Med.12:119-24). Drug resistant mutations have already been shown to developin vitro in the presence of HCV protease inhibitors (Lin, et al., 2005,In vitro studies of cross-resistance mutations against two hepatitis Cvirus serine protease inhibitors, VX-950 and BILN 2061. J. Biol. Chem.280:36784-36791; Lin, et al., 2004, In vitro resistance studies ofhepatitis C virus serine protease inhibitors, VX-950 and BILN 2061:Structural analysis indicates different resistance mechanisms. J. Biol.Chem. 279:17508-17514; Lu, et al., 2004, Antimicrob. Agents Chemother.48:2260-6; Trozzi, et al., 2003, In vitro selection and characterizationof hepatitis C virus serine protease variants resistant to anactive-site peptide inhibitor. J. Virol. 77:3669-79). Mutationsresistant to the protease inhibitor BILN 2061 have been found atpositions R155Q, A156T, and D168V/A/Y in the NS3 gene, but no mutationshave yet been observed in the NS4 region or in the protease cleavagesites. A VX-950 resistance mutation has also been found in vitro atposition A156S. Cross-resistant mutations against both VX-950 and BILN2061 have also been shown to develop in vitro at position 156 (A156V/T)(Lin, et al., 2005, supra).

Accordingly, there exists a need in identifying mutated HCVs or otherviruses that exhibit resistance to drugs or other therapies and indeveloping new viral therapeutics effective against these mutatedviruses.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides HCV variants, and relatedmethods and compositions. In particular, HCV variants and variant HCVproteases that have reduced sensitivity to one or more proteaseinhibitors such as VX-950 are provided.

In one aspect, this invention provides an isolated HCV polynucleotideencoding an HCV NS3 protease, a biologically active analog thereof, or abiologically active fragment thereof. The isolated HCV polynucleotidehas at least one codon that corresponds to codon 36, 41, 43, 54, 148,155, or 156 of a wild-type HCV polynucleotide that is mutated such thatit encodes an amino acid different from the amino acid encoded by thecorresponding codon of the wild-type HCV polynucleotide. The wild-typeHCV polynucleotide may comprise a nucleotide sequence of SEQ ID NO:1 ora portion thereof such as for example the first 543 nucleotides of SEQID NO:1. Alternatively, the wild-type HCV polynucleotide may comprise anucleotide sequence that is at least 60%, 75%, 80%, 85%, 90%, 95%, 97%,98%, 99%, or higher, identical to the sequence of SEQ ID NO:1 or aportion thereof.

In certain embodiments, the isolated HCV polynucleotide comprises acodon corresponding to codon 36 of the wild-type HCV polynucleotide, andthe codon does not encode V. In certain embodiments, the codon encodesM, L, A, or G.

In certain embodiments, the isolated HCV polynucleotide comprises acodon corresponding to codon 41 of the wild-type HCV polynucleotide, andthe codon does not encode Q. In certain embodiments, the codon encodesH.

In certain embodiments, the isolated HCV polynucleotide comprises acodon corresponding to codon 43 of the wild-type HCV polynucleotide, andthe codon does not encode F. In certain embodiments, the codon encodesS.

In certain embodiments, the isolated HCV polynucleotide comprises acodon corresponding to codon 54 of the wild-type HCV polynucleotide, andthe codon does not encode T. In certain embodiments, the codon encodes Sor A.

In certain embodiments, the isolated HCV polynucleotide comprises acodon corresponding to codon 148 of the wild-type HCV polynucleotide,and the codon does not encode G. In certain embodiments, the codonencodes E.

In certain embodiments, the isolated HCV polynucleotide comprises acodon corresponding to codon 155 of the wild-type HCV polynucleotide,and the codon does not encode R. In certain embodiments, the codonencodes K, M, S, T, G, I, or L.

In certain embodiments, the isolated HCV polynucleotide comprises acodon corresponding to codon 156 of the wild-type HCV polynucleotide,and the codon does not encode A. In certain embodiments, the codonencodes S, T, V, or I.

In certain embodiments, the isolated HCV polynucleotide comprises twocodons that correspond to any two codons selected from the groupconsisting of: codons 36, 41, 43, 54, 148, 155, and 156 of a wild-typeHCV polynucleotide, and the two codons are mutated such that eithercodon encodes an amino acid different from the amino acid encoded by thecorresponding codon of the wild-type HCV polynucleotide. For example,the isolated HCV polynucleotide comprises a codon corresponding to codon36 of the wild-type HCV polynucleotide, and the codon encodes A or M;the isolated HCV polynucleotide further comprises a codon correspondingto codon 155 of the wild-type polynucleotide, and the codon encodes K orT; alternatively, the isolated HCV polynucleotide further comprises acodon corresponding to codon 156 of the wild-type polynucleotide, andthe codon encodes T.

In certain embodiments, the isolated HCV polynucleotide comprises threecodons that correspond to any three codons selected from the groupconsisting of: codons 36, 41, 43, 54, 148, 155, and 156 of a wild-typeHCV polynucleotide, and the three codons are mutated such that each ofthe three codons encodes an amino acid different from the amino acidencoded by the corresponding codon of the wild-type HCV polynucleotide.

In certain embodiments, the isolated HCV polynucleotide comprises fourcodons corresponding to codons 36, 41, 43, 54, 148, 155, and 156 of awild-type HCV polynucleotide, and the four codons are mutated such thateach of the four codons encodes an amino acid different from the aminoacid encoded by the corresponding codon of the wild-type HCVpolynucleotide.

In further embodiments, this invention provides methods and compositionsinvolving an HCV polynucleotide of the invention. For example, anexpression system comprising the HCV polynucleotide is provided, andsuch expression system may include a vector that comprises the HCVpolynucleotide operably linked to a promoter; also provided is a hostcell transfected, transformed, or transduced with the vector.Alternatively, an expression system of the invention is based on an mRNAdisplay technology, e.g., the RNA-protein fusion technology as describedin U.S. Pat. No. 6,258,558 or the in vitro “virus” technology asdescribed in U.S. Pat. No. 6,361,943.

In another aspect, this invention provides isolated HCV variants. Anisolated HCV variant may comprise a polynucleotide encoding an HCV NS3protease, wherein at least one codon of the polynucleotide thatcorresponds to a codon selected from the group consisting of: codons 36,41, 43, 54, 148, 155, and 156 of a wild-type HCV polynucleotide ismutated such that it encodes an amino acid different from the amino acidencoded by the corresponding codon of the wild-type HCV polynucleotide.Further embodiments of the invention provide methods and compositionsinvolving the HCV variants. For example, a method is provided toidentify a compound that can inhibit replication of an HCV variant ofthe invention; a cell is provided that is infected by an HCV variant ofthe invention.

In another aspect, this invention provides isolated HCV proteases,particularly HCV NS3 proteases. An isolated HCV NS3 protease maycomprise an amino acid sequence in which the amino acid at least oneposition selected from the group consisting of: 36, 41, 43, 54, 148,155, and 156 of a wild-type HCV NS3 protease is different from the aminoacid at each corresponding position of the wild-type HCV NS3 protease.The wild type HCV NS3 protease may comprise an amino acid sequence ofSEQ ID NO:2 or a portion thereof such as for example the first 181 aminoacids of SEQ ID NO:2. The isolated HCV NS3 protease may comprise abiologically active analog or fragment of an HCV NS3 protease, forexample, the isolated HCV NS3 protease may not have the N-terminal 5,10, 15, 20, 30, 35, 40, 45, or 48 amino acids of SEQ ID NO:2.

An isolated HCV NS3 protease may also include an NS4A cofactor, such asfor example an NS4A protein as represented by the last 54 amino acids ofSEQ ID NO:2. An isolated HCV NS3 protease may be a protein complexformed by tethering an NS4A cofactor to an NS3 protease domain, forexample as described in U.S. Pat. Nos. 6,653,127 and 6,211,338.

In a further aspect, this invention provides an antibody specific to anHCV protease of the invention. The antibody may recognize an HCV NS3protease comprising an amino acid sequence in which the amino acid atleast one position selected from the group consisting of: 36, 41, 43,54, 148, 155, and 156 of a wild-type HCV NS3 protease is different fromthe amino acid at each corresponding position of the wild-type HCV NS3protease. Further embodiments of the invention provide methods andcompositions involving an anti-HCV protease antibody of the invention.For example, a diagnostic kit comprising an antibody of the invention,and a pharmaceutical compositions comprising an antibody of theinvention and a pharmaceutically acceptable carrier are provided.

In another aspect, this invention provides a nucleotide probe or primercapable of hybridizing under stringent conditions to a nucleic acidsequence of an HCV polynucleotide of the invention. Further embodimentsof the invention provide methods and compositions involving the probe orprimer. For example, a diagnostic or detection kit comprising a probe orprimer of the invention is provided, and the kit is useful in, e.g.,determining whether an HCV variant or an HCV NS3 protease of theinvention is present in a sample.

In a further aspect, this invention provides methods for evaluating drugresistance or sensitivity to a protease inhibitor of an HCV infection ina patient. Such a method may comprise collecting a biological samplefrom the HCV infected patient and evaluating or determining whether thesample comprises a nucleic acid encoding an HCV NS3 protease thatcomprises an amino acid sequence in which the amino acid at least oneposition selected from the group consisting of: 36, 41, 43, 54, 148,155, and 156 of a wild-type HCV NS3 protease is different from the aminoacid at each corresponding position of the wild-type HCV NS3 protease.The protease inhibitor may be VX-950 or another protease inhibitor.

Also provided is a method for guiding a treatment or designing atherapeutic regimen for an HCV infection in a patient. The method maycomprise evaluating drug resistance or sensitivity to a proteaseinhibitor of the patient and determining the regimen for the patientbased on the drug resistance or sensitivity. For example, if drugresistance is predicted or detected (e.g., reduced sensitivity to aprotease inhibitor such as VX-950), one or more other compounds oragents may be included in the patient's treatment plan or therapeuticregimen. The method may comprise any combination of determining thesequence (e.g., genotyping) of an HCV NS3 protease in the patient,determining the sensitivity to a protease inhibitor of an HCV NS3protease in the patient (e.g., phenotyping), or determining the viralfitness level of the patient's HCVs. The phenotyping may be carried outin a cell-free system (e.g., in vitro protease assays) as well as acell-based system (e.g., replicon assays or viral infection orreplication assays).

In another aspect, this invention provides methods for identifying acandidate compound for treating an HCV infection in a patient. Such amethod may comprise providing a sample infected with an HCV variant ofthe invention and assaying the ability of the candidate compound ininhibiting an activity of the HCV variant in the sample. The sampleinfected with an HCV variant may be obtained from a patient, such ascell or plasma samples. The sample infected with an HCV variant may alsobe cultured cells. The activity of the HCV variant may be determined byits ability to infect, replicate, and/or become released.

Alternatively, such a method may comprise providing a replicon RNAcomprising an HCV polynucleotide of the invention and determiningwhether the candidate compound inhibits replication of the replicon RNAin a suitable assay.

Another alternative method may comprise providing an isolated HCV NS3protease of invention and a protease substrate, and determining whetherthe HCV NS3 protease activity is reduced in the presence of a candidatecompound; the HCV NS3 protease and/or the protease substrate may be in acell-based system, for example expressed in cultured cells, or the HCVNS3 protease and/or the protease substrate may be in a cell-free system,for example a reaction mixture including an HCV NS3 protease and apeptide substrate. The HCV NS3 protease may be an RNA-protein fusionmolecule as described in U.S. Pat. No. 6,258,558, and such a fusionmolecules can be included in cell-free assays that evaluate proteaseactivity.

A further alternative method for evaluating a candidate compound fortreating an HCV infection in a patient may include introducing a vectorcomprising an HCV polynucleotide of the invention and an indicator geneencoding an indicator into a host cell and measuring the indicator inthe presence of the candidate compound and in the absence of thecandidate compound.

Another aspect of this invention provides a method for identifying acompound capable of rescuing the activity of VX-950 against an HCV NS3protease, for example, an HCV NS3 protease that has become resistant toVX-950. Such a compound is also termed “a secondary compound.” Themethod may comprise contacting an HCV NS3 protease of the invention witha candidate compound and assaying the ability of VX-950 to inhibit theactivity of the HCV NS3 protease. The method may also comprise the stepsof in silico modeling a variant HCV NS3 protease with reducedsensitivity to VX-950 (e.g., as determined by measuring IC₅₀ and/orK_(i)), and designing and/or selecting a compound that may rescue theactivity of VX-950.

Also provided is a method for treating an HCV infection in a patient,and the method comprises administering to the patient a pharmaceuticallyeffective amount of a secondary compound that can rescue the activity ofVX-950. The secondary compound can be administered to the patient aloneor in combination with VX-950. The secondary compound may replace VX-950in the patient's therapeutic regimen temporarily or permanently. Forexample, in a temporary replacement therapeutic regimen, VX-950 isadministered to the patient again after the compound is administered tothe patient and has rescued the activity of VX-950.

Further provided is a method for identifying a compound effective inreducing an HCV NS3 protease activity. The method may comprise obtaininga three dimensional model of an HCV NS3 protease of the invention anddesigning or selecting a compound. The method may further compriseevaluating, in silico, in vitro, and/or in vivo, the ability of thecompound to bind to or interact with the protease. The method may alsoinvolve determine whether the designed or selected compound can inhibitthe activity of an HCV NS3 protease, in particular, a variant HCV NS3protease with reduced sensitivity to a protease inhibitor such asVX-950, in a cell-free or cell-based assay. The method may further oralternatively include assaying the ability of a designed or selectedcompound to inhibit HCV replication in a cell or sample. The HCVreplication can be determined by measuring the replication of an HCVvariant of the invention or an HCV replicon of the invention.

Another aspect of this invention provides methods for eliminating orreducing HCV contamination of a biological sample, or a medical orlaboratory equipment. The method may comprise the step of contacting thebiological sample, or the medical or laboratory equipment with acompound of the invention, such as a compound identified by a methoddescribed herein.

A further aspect of this invention provides a method for treating an HCVinfection in a patient. The method may comprise administering to thepatient a pharmaceutically or therapeutically effective amount of acompound identified by a method of the invention alone or in combinationwith another anti-viral agent.

Another aspect of the invention relates to computer tools, whichprovides a machine-readable data storage medium comprising a datastorage material encoded with machine-readable data, wherein themachine-readable data comprise index values for at least two featuresassociated with an HCV variant or biological sample. The features areselected from: a) the ability to exhibit resistance for reducedsensitivity to a protease inhibitor; b) an HCV protease comprising anamino acid sequence in which the amino acid at least one positionselected from the group consisting of: 36, 41, 43, 54, 148, 155, and 156of a wild-type HCV NS3 protease is different from the amino acid at eachcorresponding position of the wild-type HCV NS3 protease; c) morbidityor recovery potential of a patient; and d) altered replication capacity(increased or decreased) of the HCV variant.

A further aspect of the invention provides a method of obtaining aprofile of HCV variants in an HCV-infected patient. The method maycomprise obtaining a sample (e.g., a plasma sample) from the patient andgenotyping and/or phenotyping an HCV protease from at least 2, 20, 50,100, 200, 500 or more HCV virions from the sample. For example, suchgenotyping may include determining the nucleotide sequence of an HCVprotease from at least 2, 20, 50, 100, 200, 500 or more HCV virions fromthe plasma sample.

In certain embodiments, the patient subjected to such profiling may havebeen treated or be selected to be treated with a protease inhibitor suchas VX-950. In certain embodiments, plasma samples are obtained from thepatient subjected to such profiling at two or more different timepoints.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a phylogenetic analysis of baseline sequences of theN terminal 543 nucleotides of the NS3 protein from untreated genotype 1HCV-infected subjects.

FIG. 2 shows the baseline IC₅₀s of Telaprevir (VX-950) for genotype 1aand 1b protease variants.

FIG. 3 illustrates the grouping of subjects based on viral response toVX-950.

FIG. 4 (in color) summarizes viral responses corresponding to mutationpatterns.

FIG. 5 shows enzymatic IC₅₀s and fold change from the reference genotype1a strain of HCV-H of protease single resistance mutants to VX-950.

FIG. 6 shows enzymatic IC₅₀s and fold change from the reference genotype1a strain of HCV-H of protease double resistance mutants to VX-950.

FIG. 7 shows the inverse correlation between resistance to VX-950 andfitness.

FIG. 8 illustrates the structure of two HCV protease inhibitors: VX-950and BILN 2061.

FIG. 9 illustrates the location of VX-950 variations in the HCV proteaseaccording to structural studies.

FIG. 10 outlines the methods for phenotypic analysis of HCV viralvariants.

FIG. 11 shows that V36 substitutions confer low-level resistance toVX-950.

FIG. 12 shows X-ray structure of the V36M variant protease.

FIG. 13 shows that V36 does not make direct contact with VX-950.

FIG. 14 shows the V36M variant in G1a with low-level resistance andbetter fitness.

FIG. 15 shows the V36A variant in G1a/b with low-level resistance andworse fitness.

FIG. 16 shows the V36G variant in G1b with low-level resistance andworse fitness.

FIG. 17 shows the V36L variant with no resistance, which is also rare inG1.

FIG. 18 also outlines the methods for phenotypic analyses of HCV viralvariants.

FIG. 19 shows that R155 substitutions confer low-level resistance toVX-950.

FIG. 20 shows the X-ray structure of the R155K variant protease.

FIG. 21 shows the computer model of VX-950 binding to the R155K variantprotease.

FIG. 22 shows that V36 or T54 substitutions confer low-level resistanceto VX-950.

FIG. 23 shows the computer model of VX-950 binding to the V36M variantprotease.

FIG. 24 shows that the V36M and R155K substitutions are additive inconferring resistance to VX-950.

FIG. 25 shows results of structural studies: (A) Superimposition of theX-ray structure of the Lys¹⁵⁵ variant and the Arg¹⁵⁵ wild-type NS3protease domain in a complex with the NS4A co-factor. The Cα atom tracesof both the wild-type (in blue) and the R155K variant (in red) proteasesare shown as lines. The residue 155 is highlighted with either ball andstick model (Arg¹⁵⁵) or Liquorice model (Lys¹⁵⁵) with nitrogens in blueand oxygens in red. (B) Superposition of side chains of Arg¹⁵⁵, Asp¹⁶⁸and Arg¹²³ in the wild type NS3-4A with that of corresponding Lys¹⁵⁵,Asp¹⁶⁸ and Arg¹²³ in the R155K variant. Three residues of the R155Kvariant protease (Arg¹²³, Asp¹⁶⁸, and Lys¹⁵⁵) are shown in the Liquoricemodel, so is the Arg¹⁵⁵ of the wild-type protease. The Arg¹²³ and Asp¹⁶⁸residues of the wild-type protease are shown as thin lines. Allnitrogens are colored in blue and oxygens in red.

FIG. 26 shows computational models of a co-complex of telaprevir withthe HCV NS3 protease domains in a complex with an NS4A cofactor. In allthree models, including the wild-type (A), R155K (B) or R155T (C)variant proteases, telaprevir is shown in a stick diagram colored inlight blue with nitrogens in blue and oxygens in red. The active siteresidues (His⁵⁷, Asp⁸¹, and Ser¹³⁹) are shown as gray sticks. The Arg¹²³and Asp¹⁶⁸ residues are colored in purple, while residue 155 side-chainis colored in yellow. The Lys¹⁵⁵ or Thr¹⁵⁵ side-chain remains in theextended conformation making minimal contacts with the P2 group oftelaprevir.

FIG. 27 shows that the VX-950 resistant replicon variants remain fullysensitive to IFN-alpha.

FIG. 28 shows that the VX-950 resistant replicon variants remain fullysensitive to Ribavirin.

FIG. 29 shows that VX-950 combination therapy suppressed emergence ofviral resistance and prevented viral breakthrough during dosing.

FIG. 30 provides summary points regarding HCV sequence diversity andresistance mutations.

FIG. 31 summarizes the mechanisms of viral variants resistance to HCVprotease inhibitors including previous studies.

FIG. 32 outlines conclusions regarding the mechanisms of viral variantsresistance to HCV protease inhibitors including previous and presentstudies.

FIG. 33 summarizes certain conclusions based on the present studies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to HCV variants. In particular, HCVvariants that exhibit resistance to a protease inhibitor are provided.Also provided are methods and compositions related to the HCV variants.The methods and compositions are useful in identifying viral variants,including variants of an HCV and other viruses, evaluating andidentifying anti-viral compounds, and developing and optimizingtherapeutics against viral infections.

The present invention is based on a study that first characterized theextent of sequence diversity within the NS3 protease domain of an HCVisolated from 34 subjects enrolled in a clinical trial, StudyVX04-950-101, before dosing with VX-950. Emergence of resistance toVX-950 in vivo was then monitored by sequence analysis of the proteaseNS3-4A region in the subjects after 14 days of dosing with VX-950. Afollow-up sample was further collected 7 to 10 days after the end ofdosing to see whether any drug-resistant mutations that developed duringdosing was maintained in the plasma after removal of VX-950. Anymutations found to have increased in the population above baseline wereconsidered potential drug resistant mutations. Because drug-resistancemutations may take some time to accumulate to a measurable level, thestudy included a new method to detect minor populations of variants(instead the dominant species in a population of wild-type viruses andviral variants), which involved obtaining sequences from many (e.g.,80-85) individual viral clones per subject per time point, so that viralvariants that may emerge in 14 days of dosing with VX-950 with asensitivity of down to about 5% of the population can be detected andidentified. Such 80/85 individual viral clones may represent up to 80/85different virions.

HCV Variants and Related Polynucleotides and Proteases

The present invention provides HCV variants. In particular embodiments,an HCV variant includes a polynucleotide sequence that encodes an HCVprotease with reduced sensitivity to a protease inhibitor (also termed“a variant HCV protease”), such as VX-950. As used herein, a wild-typeHCV refers to an HCV comprising a polynucleotide (also termed “awild-type polynucleotide”) that encodes an HCV protease with normal ordesirable sensitivity to a protease inhibitor, and in particularembodiments, the protease inhibitor is VX-950. Similarly, a wild-typeHCV protease refers to an HCV protease with normal or desirablesensitivity to a protease inhibitor, and in particular embodiments, theprotease inhibitor is VX-950.

As used here in, an HCV can be an HCV of any genotype or subtype, forexample, genotypes 1-6.

As used herein, an “NS3 protease” or an “HCV NS3 protease” refers to anHCV NS protein 3 or a portion thereof that has serine protease activity.For example, an NS3 protease can be the NS3 protein as represented bythe first 631 amino acid sequence of SEQ ID NO:2 (685 amino acids);alternatively, an NS3 protease can be a protein as represented by thefirst 181 amino acids of SEQ ID NO:2; the 181-amino acid fragment isalso referred to as the NS3 protease domain in the art. An NS3 proteasecan also be an NS3-NS4A protein complex, such as the complexes describedin U.S. Pat. Nos. 6,653,127; 6,211,338. An “NS3 protease activity” meansthe protease activity of an HCV NS protein 3 or a portion thereof in thepresence or absence of an NS4A protein or a biologically active portionthereof. An NS4A protein, such as for example as represented by the last54 amino acid sequence of SEQ ID NO:2, usually functions as a co-factorfor an NS3 protease and can form an NS3-NS4A serine protease complex; abiologically active portion of an NS4A protein refers to a fragment ofan NS4A protein that retains the NS4A protein's function as a co-factorfor an NS3 protease.

The present invention also provides isolated HCV variants, isolatedvariant HCV NS3 proteases, and isolated polynucleotide that encodes avariant HCV NS3 protease. The term “isolated” generally means separatedand/or recovered from a component of natural environment of a subjectvirus, protease, or polynucleotide.

In certain embodiments, a variant HCV protease may be a variant HCV NS3protease that comprises an amino acid sequence in which the aminoacid(s) at one or more positions from positions 36, 41, 43, 54, 148,155, or 156 of a wild-type HCV NS3 protease is(are) different from theamino acid at each corresponding position of the wild-type HCV NS3protease. The wild type HCV NS3 protease may comprise an amino acidsequence of SEQ ID NO:2 or a portion thereof such as for example thefirst 181 amino acids of SEQ ID NO:2. The isolated HCV NS3 protease maycomprise a biologically active analog or fragment of an HCV NS3protease, for example, the isolated HCV NS3 protease may not have theN-terminal 5, 10, 15, 20, 30, 35, 40, 45, or 48 amino acids of SEQ IDNO:2.

Examples of amino acid substitutions or mutations at various positionsof a variant HCV NS3 protease are shown in Tables 1-4. The Tables,Figures, and Examples herein also provide various data obtained withvariant HCV NS3 proteases or HCV viral variants as compared to wild-typeHCV NS3 proteases or wild-type HCVs.

Biologically active fragments or analogs of a variant HCV NS3 proteaseof the invention are also provided. Bartenschlager et al. (1994, J.Virology 68: 5045-55) described various fragments of HCV NS3 proteins,for example, the deletion of N-terminal 7 or 23 residues abolishedcleavage at NS4B/5A site, but no effect on other cleave sites subjectedto the NS3 protease activity; and the deletion of N-terminal 39 residuesabolished cleavage at NS4B/5A and NS5A/5B sites and decreased the NS3protease activity on the NS4A/4B site. Failla et al. (1995, J. Virology69: 1769-77) described that the deletion of N-terminal 10 residues of awild-type NS3 protein had no effect on the NS3 protease activity, thedeletion of N-terminal 15 or 28 residues resulted in a NS3 protein withpartial protease activity (normal cleavage at NS5A/5B, but lower atNS4A/4B and NS4B/5A sites), the deletion of N-terminal 49 residuesresulted in a completely inactive NS3 protease, and the deletion ofC-terminal 10 residues of the NS3 protease domain in the NS3 proteinalso resulted in a completely inactive NS3 proteases.

Expression systems are provided, for example, to make the variant HCVproteases of the invention. An expression system may include anexpression vector that comprises an HCV polynucleotide of the invention.Suitable prokaryotic or eukaryotic vectors (e.g., expression vectors)comprising an HCV polynucleotide (or “nucleic acid,” usedinterchangeably herein) of the invention can be introduced into asuitable host cell by an appropriate method (e.g., transformation,transfection, electroporation, infection), such that the polynucleotideis operably linked to one or more expression control elements (e.g., inthe vector or integrated into the host cell genome). For production,host cells can be maintained under conditions suitable for expression(e.g., in the presence of inducer, suitable media supplemented withappropriate salts, growth factors, antibiotic, nutritional supplements,etc.), whereby the encoded polypeptide is produced. If desired, theencoded protein can be recovered and/or isolated (e.g., from the hostcells or medium). It will be appreciated that the method of productionencompasses expression in a host cell of a transgenic animal (see e.g.,WO 92/03918). An expression system may be based on a cell-free systemsuch as the RNA-protein fusion technology described in U.S. Pat. No.6,258,558 or the in vitro “virus” described in U.S. Pat. No. 6,361,943.Ribosome display method can also be used, such as the method describedin U.S. Pat. No. 5,843,701.

Various assays are provided, for example, assays suitable forphenotyping HCVs. The assays may be directed to measuring a viralactivity (e.g., infection, replication, and/or release of viralparticles) or an enzymatic activity (e.g. protease activity). Viralactivity assays may employ cells or samples infected with a virus orviral variant of which the activity is to be measured. The cells orsamples may be obtained from a patient such as a human patient.Alternatively, the cells or samples may be cultured and infected with avirus or viral variant in vitro. Viral activity assays may employ areplicon-based system, such as the replicon-based assays described inTrozzi et al. (13) and U.S. patent application publication No.20050136400.

Enzymatic activity can be determined in cell-free or cell-based systemswhich generally include the enzyme of interest or a biologically activefragment or analog thereof and a substrate for the enzyme of interest.For example, U.S. patent application publication No. 20030162169describes a surrogate cell-based system and method for assaying theactivity of HCV NS3 protease. Trozzi et al. (13) describes an in vitro,cell-free protease assay that employs peptide substrates and HPLCsystems.

The present invention takes advantage of the fact that thethree-dimensional structure of NS3/4A protease has been resolved (seee.g., WO 98/11134). A three dimensional model of the variant protease ofthe invention can be obtained; compounds are designed or selected, forexample based on their ability to interact with the three-dimensionalstructure of the variant protease, and the ability to bind to orinteract with the protease is evaluated by modeling in silico and can befurther evaluated by in vitro or in vivo assays.

The compound may be one identified from a combinatorial chemical libraryor prepared through rational drug design. In exemplary embodiments, thecompound is a compound prepared through rational drug design and derivedfrom the structure of a known protease inhibitor such as VX-950.Rational drug design also may be combined with a systematic method oflarge-scale screening experiments where potential protease inhibitordrug targets are tested with compounds from combinatorial libraries.Rational drug design is a focused approach, which uses information aboutthe structure of a drug receptor or one of its natural ligands toidentify or create candidate drugs. The three-dimensional structure of aprotein can be determined using methods such as X-ray crystallography ornuclear magnetic resonance spectroscopy. In the present invention, thethree dimensional structure of a variant HCV NS3 protease that containsone or more of the mutations of residues 36, 41, 43, 54, 148, 155, or156 may now readily be determined using routine X-ray crystallographicand/or NMR spectroscopy techniques. Rational drug design also may becombined with a systematic method of large-scale screening experimentswhere potential protease inhibitor drug targets are tested withcompounds from combinatorial libraries. Computer programs can be devisedto search through databases containing the structures of many differentchemical compounds. The computer can select those compounds that aremost likely to interact with the variant HCV NS3 proteases, and suchidentified compound can be tested in assays (e.g., viral or enzymaticassays) suitable for evaluating protease inhibitors.

In certain embodiments, the identified compound is formulated into acomposition comprising the compound and a pharmaceutically acceptablecarrier, adjuvant or vehicle. Preferably the composition contains thecompound in an amount effective to reduce the activity of an HCV NS3serine protease. Even more preferably the composition is formulated foradministration to a patient. The compositions also may comprise anadditional agent selected from an immunomodulatory agent; an anti-viralagent; a second inhibitor of HCV protease; an inhibitor of anothertarget in the HCV life cycle; a cytochrome P-450 inhibitor; orcombinations thereof. The various compositions are described in greaterdetails below.

In another aspect, the present invention provides antibodies that arespecific to an HCV protease, in particular, an HCV NS3 protease with oneor more amino acids altered as compared to a wild type HCV NS3 protease.The term “antibody” is used in the broadest sense and specificallycovers, without limitation, intact monoclonal antibodies, polyclonalantibodies, chimeric antibodies, multispecific antibodies (e.g.,bispecific antibodies) formed from at least two intact antibodies, andantibody fragments, so long as they exhibit the desired biologicalactivity. The term “immunoglobulin” includes a variety of structurallyrelated proteins that are not necessarily antibodies.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen-binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.,8 (10): 1057-1062 (1995)); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of an antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains that enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore, eds. (Springer-Verlag: New York, 1994), pp.269-315.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90: 6444-6448 (1993).

An antibody against a variant HCV protease may be developed from a knownantibody against an HCV NS3 protein, for example through molecularevolution. U.S. patent application publication No. 20040214994 describesan human recombinant antibody against the HCV NS3 protein. Amino acidsequence variants of are prepared by introducing appropriate nucleotidechanges into the DNA of a known antibody, or by peptide synthesis. Suchvariants include, for example, deletions from, and/or insertions intoand/or substitutions of, residues within the amino acid sequences of theknown antibody. Any combination of deletion, insertion, and substitutionis made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics. The amino acid changesalso may alter post-translational processes of the antibody, such aschanging the number or position of glycosylation sites.

An antibody of the invention may have diagnostic as well as therapeuticapplications. In certain embodiments, an antibody of the invention islabeled. The various antibodies of the present disclosure can be used todetect or measure the expression of a variant HCV NS3 protease, andtherefore, they are also useful in applications such as cell sorting andimaging (e.g., flow cytometry, and fluorescence activated cell sorting),for diagnostic or research purposes. As used herein, the terms “label”or “labeled” refers to incorporation of another molecule in theantibody. In one embodiment, the label is a detectable marker, e.g.,incorporation of a radiolabeled amino acid or attachment to apolypeptide of biotinyl moieties that can be detected by marked avidin(e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or colorimetric methods). Inanother embodiment, the label or marker can be therapeutic, e.g., a drugconjugate or toxin. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (e.g., leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags),magnetic agents, such as gadolinium chelates, toxins such as pertussistoxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. In some embodiments, labels are attached by spacerarms of various lengths to reduce potential steric hindrance.

In certain aspects, kits for use in detecting the presence of an HCVviral, a variant HCV NS3 polynucleotide, or a variant HCV protease in abiological sample can also be prepared. Such kits may include anantibody that recognizes a variant HCV NS3 protease of the invention, aswell as one or more ancillary reagents suitable for detecting thepresence of a complex between the antibody and the variant protease or aportion thereof. Alternatively, such kits may include a probe or primerof the invention, such a probe or primer can hybridize with a variantHCV NS3 polynucleotide of the invention under stringent conditions. Aprobe or primer of the invention may be suitable for PCR or RT-PCR thatcan be employed to detect a subject of interest. Alternatively, suchkits may be based on PCR or non-PCR based HCV diagnostic kits availablecommercially, e.g., Roche Cobas Amplicor system and Bayer Versantsystem, including RNA 3.0 assay (bDNA) and RNA Qualitative Assay (TMA).The AMPLICOR HCV MONITOR® Test, v2.0 is an in vitro nucleic acidamplification test for the quantification of HCV RNA in human serum orplasma. The VERSANT® HCV RNA 3.0 Assay (bDNA) is a viral load assay thathas been proven to reliably detect a 2 log 10 drop. The VERSANT® HCV RNAQualitative Assay is based on state-of-the-art Transcription-MediatedAmplification (TMA) technology.

Pharmaceutical Compositions and Formulations

Another aspect of the invention provides pharmaceutical compositions orformulations including a compound of the invention, for example, asecondary compound that is identified as being able to rescue theactivity of VX-950, or a compound that is identified as effectiveagainst an HCV variant (e.g., capable of reducing replication of theviral variant) and/or a variant HCV NS3 protease (e.g., capable ofreducing the enzymatic activity of the variant protease).

Another aspect of the invention provides uses of a compound of theinvention in the manufacture of a medicament, such as a medicament fortreating an HCV infection in a patient.

Another aspect of the invention provides methods for treating an HCVinfection in a patient. Such methods generally comprise administering tothe patient a pharmaceutically or therapeutically effective amount of acompound of the invention alone or in combination (sequentially orcontemporaneously) with another anti-viral agent. “Effective amount” ofa compound or agent generally refers to those amounts effective toreproducibly reduce HCV NS3 protease expression or activity, HCVproduction, replication, or virulence, HCV infection, or produce anamelioration or alleviation of one or more of the symptoms of HCVinfection in comparison to the levels of these parameters in the absenceof such a compound or agent.

In another aspect, the methods and compositions of this inventioninclude a protease inhibitor (e.g., VX-950) and another anti-viralagent, preferably an anti-HCV agent. Combination therapy targeting HCVis also described in U.S. Pat. Nos. 6,924,270; 6,849,254.

Another anti-viral agent may also be a protease inhibitor, particularlyan HCV protease inhibitor. HCV protease inhibitors known in the artinclude VX-950 (FIG. 8), BILN 2061 (FIG. 8, see also PCT Publication No.WO 00/59929; U.S. Pat. No. 6,608,027), compound 1 (13), Inhibitors A, B,and C(PCT Publication No. WO 04/039970). Potential HCV proteaseinhibitors have also been described in PCT and U.S. patent applicationpublication Nos. WO 97/43310, US 20020016294, WO 01/81325, WO 02/08198,WO 01/77113, WO 02/08187, WO 02/08256, WO 02/08244, WO 03/006490, WO01/74768, WO 99/50230, WO 98/17679, WO 02/48157, US 20020177725, WO02/060926, US 20030008828, WO 02/48116, WO 01/64678, WO 01/07407, WO98/46630, WO 00/59929, WO 99/07733, WO 00/09588, US 20020016442, WO00/09543, WO 99/07734, US 20020032175, US 20050080017, WO 98/22496, WO02/079234, WO 00/31129, WO 99/38888, WO 99/64442, WO 2004072243, and WO02/18369, and U.S. Pat. Nos. 6,018,020; 6,265,380; 6,608,027; 5,866,684;M. Llinas-Brunet et al., Bioorg. Med. Chem. Lett., 8, pp. 1713-18(1998); W. Han et al., Bioorg. Med. Chem. Lett., 10, 711-13 (2000); R.Dunsdon et al., Bioorg. Med. Chem. Lett., 10, pp. 1571-79 (2000); M.Llinas-Brunet et al., Bioorg. Med. Chem. Lett., 10, pp. 2267-70 (2000);and S. LaPlante et al., Bioorg. Med. Chem. Lett., 10, pp. 2271-74(2000). A number of NS3 protease inhibitors have also been developed bySchering Corp., Schering A. G., and other companies, and they aredescribed in U.S. patent application publication Nos. 20050249702;20050153900; 20050245458; 20050222047; 20050209164; 20050197301;20050176648; 20050164921; 20050119168; 20050085425; 20050059606;20030207861; 20020147139; 20050143439; 20050059606; 20050107304;20050090450; 20040147483; 20040142876; 20040077600; 20040018986;20030236242; 20030216325; 20030207861; U.S. Pat. Nos. 6,962,932;6,914,122; 6,911,428; 6,846,802; 6,838,475.

Anti-viral agents may also include, but are not limited to,immunomodulatory agents, such as alpha-, beta-, and gamma-interferons,pegylated derivatized interferon-alpha compounds, and thymosin; otheranti-viral agents, such as ribavirin, amantadine, and telbivudine; otherinhibitors of hepatitis C proteases (NS2-NS3 inhibitors and NS3-NS4Ainhibitors); inhibitors of other targets in the HCV life cycle,including helicase and polymerase inhibitors; inhibitors of internalribosome entry; broad-spectrum viral inhibitors, such as IMPDHinhibitors (e.g., compounds of U.S. Pat. Nos. 5,807,876, 6,498,178,6,344,465, 6,054,472, WO 97/40028, WO 98/40381, WO 00/56331, andmycophenolic acid and derivatives thereof, and including, but notlimited to VX-497, VX-148, and/or VX-944); or combinations of any of theabove. See also W. Markland et al., Antimicrobial & AntiviralChemotherapy, 44, p. 859 (2000) and U.S. Pat. No. 6,541,496.

The following definitions are used herein:

“Peg-Intron” means PEG-Intron®, peginteferon alfa-2b, available fromSchering Corporation, Kenilworth, N.J.; “Intron” means Intron-A®,interferon alpha-2b available from Schering Corporation, Kenilworth,N.J.; “ribavirin” means ribavirin(1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available fromICN Pharmaceuticals, Inc., Costa Mesa, Calif.; described in the MerckIndex, entry 8365, Twelfth Edition; also available as Rebetol® fromSchering Corporation, Kenilworth, N.J., or as Copegus® from Hoffmann-LaRoche, Nutley, N.J.; “Pagasys” means Pegasys®, peg-interferon alfa-2aavailable Hoffmann-La Roche, Nutley, N.J.; “Roferon” means Roferon®,recombinant interferon alpha-2a available from Hoffmann-La Roche,Nutley, N.J.; “Berofor” means Berofor®, interferon alpha-2 availablefrom Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, Conn.;Sumiferon®, a purified blend of natural alpha interferons such asSumiferon available from Sumitomo, Japan; Wellferon®, interferon alphan1 available from Glaxo Wellcome Ltd., Great Britain; Alferon®, amixture of natural alpha interferons made by Interferon Sciences, andavailable from Purdue Frederick Co., CT.

The term “interferon” as used herein means a member of a family ofhighly homologous species-specific proteins that inhibit viralreplication and cellular proliferation, and modulate immune response,such as interferon alpha, interferon beta, or interferon gamma. TheMerck Index, entry 5015, Twelfth Edition. According to one embodiment ofthe present invention, the interferon is alpha-interferon. According toanother embodiment, a therapeutic combination of the present inventionutilizes natural alpha interferon 2a. Alternatively, the therapeuticcombination of the present invention utilizes natural alpha interferon2b. In another embodiment, the therapeutic combination of the presentinvention utilizes recombinant alpha interferon 2a or 2b. In yet anotherembodiment, the interferon is pegylated alpha interferon 2a or 2b.Interferons suitable for the present invention include: (a) Intron(interferon-alpha 2B, Schering Plough), (b) Peg-Intron, (c) Pegasys, (d)Roferon, (e) Berofor, (f) Sumiferon, (g) Wellferon, (h) consensus alphainterferon available from Amgen, Inc., Newbury Park, Calif., (i)Alferon; (j) Viraferon®; (k) Infergen®.

A protease inhibitor can be administered orally, whereas Interferon isnot typically administered orally. Nevertheless, nothing herein limitsthe methods or combinations of this invention to any specific dosageforms or regime. Thus, each component of a combination according to thisinvention may be administered separately, together, sequentially orsimultaneously, or in any combination thereof.

In one embodiment, the protease inhibitor and interferon areadministered in separate dosage forms. In one embodiment, any additionalagent is administered as part of a single dosage form with the proteaseinhibitor or as a separate dosage form. As this invention involves acombination of compounds and/or agents, the specific amounts of eachcompound or agent may be dependent on the specific amounts of each othercompound in the combination. Dosages of interferon are typicallymeasured in IU (e.g., about 4 million IU to about 12 million IU).

Accordingly, agents (whether acting as an immunomodulatory agent orotherwise) that may be used in combination with a compound of thisinvention include, but are not limited to, interferon-alpha 2B (IntronA, Schering Plough); Rebatron (Schering Plough, Inteferon-alpha2B+Ribavirin); pegylated interferon alpha (Reddy, K. R. et al. “Efficacyand Safety of Pegylated (40-kd) interferon alpha-2a compared withinterferon alpha-2a in noncirrhotic patients with chronic hepatitis C,”Hepatology, 33, pp. 433-438 (2001); consensus interferon (Kao, J. H., etal., “Efficacy of Consensus Interferon in the Treatment of ChronicHepatitis,” J. Gastroenterol. Hepatol. 15, pp. 1418-1423 (2000),interferon-alpha 2A (Roferon A; Roche), lymphoblastoid or “natural”interferon; interferon tau (Clayette, P. et al., “IFN-tau, A NewInterferon Type I with Antiretroviral activity,” Pathol. Biol. (Paris)47, pp. 553-559 (1999); interleukin 2 (Davis, G. L. et al., “FutureOptions for the Management of Hepatitis C,” Seminars in Liver Disease,19, pp. 103-112 (1999); Interleukin 6 (Davis, G. L. et al., supra;interleukin 12 (Davis, G. L. et al., supra; Ribavirin; and compoundsthat enhance the development of type 1 helper T cell response (Davis, G.L., et al., supra. Interferons may ameliorate viral infections byexerting direct antiviral effects and/or by modifying the immuneresponse to infection. The antiviral effects of interferons are oftenmediated through inhibition of viral penetration or uncoating, synthesisof viral RNA, translation of viral proteins, and/or viral assembly andrelease.

Compounds that stimulate the synthesis of interferon in cells(Tazulakhova, E. B. et al., “Russian Experience in Screening, analysis,and Clinical Application of Novel Interferon Inducers,” J. InterferonCytokine Res., 21 pp. 65-73) include, but are not limited to, doublestranded RNA, alone or in combination with tobramycin, and Imiquimod (3MPharmaceuticals; Sauder, D. N., “Immunomodulatory and PharmacologicProperties of Imiquimod,” J. Am. Acad. Dermatol., 43 pp. S6-11 (2000).

Other non-immunomodulatory or immunomodulatory compounds may be used incombination with a compound of this invention including, but not limitedto, those specified in WO 02/18369, which is incorporated herein byreference (see, e.g., page 273, lines 9-22 and page 274, line 4 to page276, line 11, which is incorporated herein by reference in itsentirety).

Compounds that stimulate the synthesis of interferon in cells(Tazulakhova et al., J. Interferon Cytokine Res. 21, 65-73)) include,but are not limited to, double stranded RNA, alone or in combinationwith tobramycin and Imiquimod (3M Pharmaceuticals) (Sauder, J. Am. Arad.Dermatol. 43, S6-11 (2000)).

Other compounds known to have, or that may have, HCV antiviral activityinclude, but are not limited to, Ribavirin (ICN Pharmaceuticals);inosine 5′-monophosphate dehydrogenase inhibitors (VX-497 formulaprovided herein); amantadine and rimantadine (Younossi et al., InSeminars in Liver Disease 19, 95-102 (1999)); LY217896 (U.S. Pat. No.4,835,168) (Colacino, et al., Antimicrobial Agents & Chemotherapy 34,2156-2163 (1990)); and9-Hydroxyimino-6-methoxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydro-phena-nthrene-1-carboxylicacid methyl ester; 6-Methoxy-1,4adimethyl-9-(4-methyl-piperazin-1-ylimino)-1,2,3,4,4a,9,10,10a-octahydro-p-henanthrene-1carboxylicacid methyl ester-hydrochloride;1-(2-Chloro-phenyl)-3-(2,2-Biphenyl-ethyl)-urea (U.S. Pat. No.6,127,422).

Formulations, doses, and routes of administration for the foregoingmolecules are either taught in the references cited below, or arewell-known in the art as disclosed, for example, in F. G. Hayden, inGoodman & Gilman's The Pharmacological Basis of Therapeutics, NinthEdition, Hardman et al., Eds., McGraw-Hill, New York (1996), Chapter 50,pp. 1191-1223, and the references cited therein. Alternatively, once acompound that exhibits HCV antiviral activity, particularly antiviralactivity against a drug-resistant strain of HCV, has been identified, apharmaceutically effective amount of that compound can be determinedusing techniques that are well-known to the skilled artisan. Note, forexample, Benet et al., in Goodman & Gilman's The Pharmacological Basisof Therapeutics, Ninth Edition, Hardman et al., Eds., McGraw-Hill, NewYork (1996), Chapter 1, pp. 3-27, and the references cited therein.Thus, the appropriate formulations, dose(s) range, and dosing regimens,of such a compound can be easily determined by routine methods.

The compositions related to combination therapies of the presentinvention can be provided to a cell or cells, or to a human patient,either in separate pharmaceutically acceptable formulations administeredsimultaneously or sequentially, formulations containing more than onetherapeutic agent, or by an assortment of single agent and multipleagent formulations. Regardless of the route of administration, thesedrug combinations form an anti-HCV effective amount of components of thepharmaceutically acceptable formulations.

A large number of other immunomodulators and immunostimulants that canbe used in the methods of the present invention are currently availableand include: AA-2G; adamantylamide dipeptide; adenosine deaminase, Enzonadjuvant, Alliance; adjuvants, Ribi; adjuvants, Vaxcel; Adjuvax;agelasphin-11; AIDS therapy, Chiron; algal glucan, SRI; alganunulin,Anutech; Anginlyc; anticellular factors, Yeda; Anticort; antigastrin-17immunogen, Ap; antigen delivery system, Vac; antigen formulation, IDBC;antiGnRH immunogen, Aphton; Antiherpin; Arbidol; azarole; Bay-q-8939;Bay-r-1005; BCH-1393; Betafectin; Biostim; BL-001; BL-009; Broncostat;Cantastim; CDRI-84-246; cefodizime; chemokine inhibitors, ICOS; CMVpeptides, City of Hope; CN-5888; cytokine-releasing agent, St; DHEAS,Paradigm; DISC TA-HSV; J07B; I01A; I01Z; ditiocarb sodium; ECA-10-142;ELS-1; endotoxin, Novartis; FCE-20696; FCE-24089; FCE-24578; FLT-3ligand, Immunex; FR-900483; FR-900494; FR-901235; FTS-Zn; G-proteins,Cadus; gludapcin; glutaurine; glycophosphopeptical; GM-2; GM-53; GMDP;growth factor vaccine, EntreM; H-BIG, NABI; H-CIG, NABI; HAB-439;Helicobacter pylori vaccine; herpes-specific immune factor; HIV therapy,United Biomed; HyperGAM+CF; ImmuMax; Immun BCG; immune therapy,Connective; immunomodulator, Evans; immunomodulators, Novacell; imreg-1;imreg-2; Indomune; inosine pranobex; interferon, Dong-A (alpha2);interferon, Genentech (gamma); interferon, Novartis (alpha);interleukin-12, Genetics Ins; interleukin-15, Immunex; interleukin-16,Research Cor; ISCAR-1; J005X; L-644257; licomarasminic acid; LipoTher;LK-409, LK-410; LP-2307; LT (R1926); LW-50020; MAF, Shionogi; MDPderivatives, Merck; met-enkephalin, TNI; methylfurylbutyrolactones;MIMP; mirimostim; mixed bacterial vaccine, Tem, MM-1; moniliastat; MPLA,Ribi; MS-705; murabutide; marabutide, Vacsyn; muramyl dipeptidederivative; muramyl peptide derivatives myelopid; -563; NACOS-6; NH-765;NISV, Proteus; NPT-16416; NT-002; PA-485; PEFA-814; peptides, Scios;peptidoglycan, Pliva; Perthon, Advanced Plant; PGM derivative, Pliva;Pharmaprojects No. 1099; No. 1426; No. 1549; No. 1585; No. 1607; No.1710; No. 1779; No. 2002; No. 2060; No. 2795; No. 3088; No. 3111; No.3345; No. 3467; No. 3668; No. 3998; No. 3999; No. 4089; No. 4188; No.4451; No. 4500; No. 4689; No. 4833; No. 494; No. 5217; No. 530;pidotimod; pimelautide; pinafide; PMD-589; podophyllotoxin, Conpharm;POL-509; poly-ICLC; poly-ICLC, Yamasa Shoyu; PolyA-PolyU; PolysaccharideA; protein A, Berlux Bioscience; PS34W0; Pseudomonas MAbs, Teijin;Psomaglobin; PTL-78419; Pyrexol; pyriferone; Retrogen; Retropep; RG-003;Rhinostat; rifamaxil; RM-06; Rollin; romurtide; RU-40555; RU-41821;Rubella antibodies, ResCo; S-27649; SB-73; SDZ-280-636; SDZ-MRL953;SK&F-107647; SL04; SL05; SM-4333; Solutein; SRI-62-834; SRL-172; ST-570;ST-789; staphage lysate; Stimulon; suppressin; T-150R1; T-LCEF;tabilautide; temurtide; Theradigm-HBV; Theradigm-HBV; Theradigm-HSV;THF, Pharm & Upjohn; THF, Yeda; thymalfasin; thymic hormone fractions;thymocartin; thymolymphotropin; thymopentin; thymopentin analogues;thymopentin, Peptech; thymosin fraction 5, Alpha; thymostimulin;thymotrinan; TMD-232; TO-115; transfer factor, Viragen; tuftsin, Selavo;ubenimex; Ulsastat; ANGG−; CD-4+; Collag+; COLSF+; COM+; DA-A+; GAST−;GF-TH+; GP-120−; IF+; IF-A+; IF-A-2+; IF-B+; IF-G+; IF-G-1B+; IL-2+;IL-12+; IL-15+; IM+; LHRH−; LIPCOR+L LYM-B+; LYM-NK+; LYM-T+; OPI+;PEP+; PHG-MA+; RNA-SYN−; SY-CW−; TH-A-I+; TH-5+; TNF+; UN.

Representative nucleoside and nucleotide compounds useful in the presentinvention include, but are not limited to:(+)-cis-5-fluoro-1-[2-(hydroxymethyl)-[1,3-oxathiolan-5yl]cytosine;(−)-2′-deoxy-3′-thiocytidine-5′-triphospbate (3TC);(−)-cis-5-fluoro-1-[2(hydroxy-methyl)-[1,3-oxathiolan-5-yl]cytosine(FTC); (−)2′,3′, dideoxy-3′-thiacytidine[(−)-SddC];1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC);1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)-5-iodocytosinetriphosphate (FIACTP);1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)-5-m-ethyluracil (FMAU);1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide;2′,3′-dideoxy-3′-fluoro-5-methyl-dexocytidine (FddMeCyt);2′,3′-dideoxy-3′-chloro-5-methyl-dexocytidine (ClddMeCyt);2′,3′-dideoxy-3′-amino-5-methyl-dexocytidine (AddMeCyt);2′,3′-dideoxy-3′-fluoro-5-methyl-cytidine (FddMeCyt);2′,3′-dideoxy-3′-chloro-5-methyl-cytidine (ClddMeCyt);2′,3′-dideoxy-3′-amino-5-methyl-cytidine (AddMeCyt);2′,3′-dideoxy-3′-fluorothymidine (FddThd);2′,3′-dideoxy-beta-L-5-fluoroc-ytidine (beta-L-FddC)2′,3′-dideoxy-beta-L-5-thiacytidine; 2′,3′-dideoxy-beta-L-5-cytidine(beta-L-ddC); 9-(1,3-dihydroxy-2-propoxym-ethyl) guanine;2′-deoxy-3′-thia-5-fluorocytosine; 3′-amino-5-methyl-dexoc-ytidine(AddMeCyt); 2-amino-1,9-[(2-hydroxymethyl-1-(hydroxymethyl)ethoxy]methyl]-6H-purin-6-one (gancyclovir);2-[2-(2-amino-9H-purin-9y)et-hyl)-1,3-propandil diacetate(famciclovir);2-amino-1,9-dihydro-9-[(2-hydro-xy-ethoxy)methyl]6H-purin-6-one(acyclovir); 9-(4-hydroxy-3-hydroxymethyl-but-1-yl)guanine(penciclovir); 9-(4-hydroxy-3-hydroxymethyl-but-1-yl)-6-deoxy-guaninediacetate(famciclovir); 3′-azido-3′-deoxythymidine (AZT);3′-chloro-5-methyl-dexocytidine (ClddMeCyt);9-(2-phosphonyl-methoxyethyl-)-2′,6′-diaminopurine-2′,3′-dideoxyriboside;9-(2-phosphonylmethoxyethyl)-adenine (PMEA); acyclovir triphosphate(ACVTP); D-carbocyclic-2′-deoxyguan-osine (CdG); dideoxy-cytidine;dideoxy-cytosine (ddC); dideoxy-guanine (ddG); dideoxy-inosine (ddI);E-5-(2-bromovinyl)-2′-deoxyuridine triphosphate;fluoro-arabinofuranosyl-iodouracil;1-(2′-deoxy-2′-fluoro-1-beta-D-arabinofuranosyl)-5-iodo-uracil (FIAU);stavudine; 9-beta-D-arabinofuranosyl-9H-purine-6-amine monohydrate(Ara-A); 9-beta-D-arabinofuranosyl-9H-purine-6-amine-5′-monophosphatemonohydrate (Ara-AMP); 2-deoxy-3′-thia-5-fluorocytidine;2′,3′-dideoxy-guanine; and 2′,3′-dideoxy-guanosine.

Synthetic methods for the preparation of nucleosides and nucleotidesuseful in the present invention are well known in the art as disclosedin Acta Biochim Pol., 43, 25-36 (1996); Swed. Nucleosides Nucleotides15, 361-378 (1996); Synthesis 12, 1465-1479 (1995); Carbohyd. Chem. 27,242-276 (1995); Chena Nucleosides Nucleotides 3, 421-535 (1994); Ann.Reports in Med. Chena, Academic Press; and Exp. Opin. Invest. Drugs 4,95-115 (1995).

The chemical reactions described in the references cited above aregenerally disclosed in terms of their broadest application to thepreparation of the compounds of this invention. Occasionally, thereactions may not be applicable as described to each compound includedwithin the scope of compounds disclosed herein. The compounds for whichthis occurs will be readily recognized by those skilled in the art. Inall such cases, either the reactions can be successfully performed byconventional modifications known to those skilled in the art, e.g., byappropriate protection of interfering groups, by changing to alternativeconventional reagents, by routine modification of reaction conditions,and the like, or other reactions disclosed herein or otherwiseconventional will be applicable to the preparation of the correspondingcompounds of this invention. In all preparative methods, all startingmaterials are known or readily preparable from known starting materials.

While nucleoside analogs are generally employed as anti-viral agents asis, nucleotides (nucleoside phosphates) sometimes have to be convertedto nucleosides in order to facilitate their transport across cellmembranes. An example of a chemically modified nucleotide capable ofentering cells is S-1-3-hydroxy-2-phosphonylmethoxypropyl cytosine(HPMPC, Gilead Sciences). Nucleoside and nucleotide compounds used inthis invention that are acids can form salts. Examples include saltswith alkali metals or alkaline earth metals, such as sodium, potassium,calcium, or magnesium, or with organic bases or basic quaternaryammonium salts.

The skilled artisan may also chose to administer a cytochrome P450monooxygenase inhibitor. Such inhibitors may be useful in increasingliver concentrations and/or increasing blood levels of compounds thatare inhibited by cytochrome P450. For an embodiment of this inventionthat involves a CYP inhibitor, any CYP inhibitor that improves thepharmacokinetics of the relevant NS3/4A protease may be included in acomposition and/or used in a method of this invention. These CYPinhibitors include, but are not limited to, ritonavir (WO 94/14436),ketoconazole, troleandomycin, 4-methylpyrazole, cyclosporin,clomethiazole, cimetidine, itraconazole, fluconazole, miconazole,fluvoxamine, fluoxetine, nefazodone, sertraline, indinavir, nelfinavir,amprenavir, fosamprenavir, saquinavir, lopinavir, delavirdine,erythromycin, VX-944, and VX-497. Preferred CYP inhibitors includeritonavir, ketoconazole, troleandomycin, 4-methylpyrazole, cyclosporin,and clomethiazole. For preferred dosage forms of ritonavir, see U.S.Pat. No. 6,037,157, and the documents cited therein: U.S. Pat. No.5,484,801, U.S. application Ser. No. 08/402,690, and InternationalApplications WO 95/07696 and WO 95/09614).

Methods for measuring the ability of a compound to inhibit cytochromeP50 monooxygenase activity are known (see U.S. Pat. No. 6,037,157 andYun, et al. Drug Metabolism & Disposition, vol. 21, pp. 403-407 (1993).

Immunomodulators, immunostimulants and other agents useful in thecombination therapy methods of the present invention can be administeredin amounts lower than those conventional in the art. For example,interferon alpha is typically administered to humans for the treatmentof HCV infections in an amount of from about 1×10⁶ units/person threetimes per week to about 10.times.106 units/person three times per week(Simon et al., Hepatology 25: 445-448 (1997)). In the methods andcompositions of the present invention, this dose can be in the range offrom about 0.1×10⁶ units/person three times per week to about 7.5×10⁶units/person three times per week; more preferably from about 0.5×10⁶units/person three times per week to about 5×10⁶ units/person threetimes per week; most preferably from about 1×10⁶ units/person threetimes per week to about 3×10⁶ units/person three times per week. Due tothe enhanced hepatitis C virus antiviral effectiveness ofimmunomodulators, immunostimulants or other anti-HCV agent in thepresence of the HCV serine protease inhibitors of the present invention,reduced amounts of these immunomodulators/immunostimulants can beemployed in the treatment methods and compositions contemplated herein.Similarly, due to the enhanced hepatitis C virus antiviral effectivenessof the present HCV serine protease inhibitors in the presence ofimmunomodulators and immunostimulants, reduced amounts of these HCVserine protease inhibitors can be employed in the methods andcompositions contemplated herein. Such reduced amounts can be determinedby routine monitoring of hepatitis C virus titers in infected patientsundergoing therapy. This can be carried out by, for example, monitoringHCV RNA in patients' serum by slot-blot, dot-blot, or RT-PCR techniques,or by measurement of HCV surface or other antigens. Patients can besimilarly monitored during combination therapy employing the HCV serineprotease inhibitors disclosed herein and other compounds having anti-HCVactivity, for example nucleoside and/or nucleotide anti-viral agents, todetermine the lowest effective doses of each when used in combination.

In the methods of combination therapy disclosed herein, nucleoside ornucleotide antiviral compounds, or mixtures thereof, can be administeredto humans in an amount in the range of from about 0.1 mg/person/day toabout 500 mg/person/day; preferably from about 10 mg/person/day to about300 mg/person/day; more preferably from about 25 mg/person/day to about200 mg/person/day; even more preferably from about 50 mg/person/day toabout 150 mg/person/day; and most preferably in the range of from about1 mg/person/day to about 50 mg/person/day.

Doses of compounds can be administered to a patient in a single dose orin proportionate doses. In the latter case, dosage unit compositions cancontain such amounts of submultiples thereof to make up the daily dose.Multiple doses per day can also increase the total daily dose shouldthis be desired by the person prescribing the drug.

The regimen for treating a patient suffering from a HCV infection withthe compounds and/or compositions of the present invention is selectedin accordance with a variety of factors, including the age, weight, sex,diet, and medical condition of the patient, the severity of theinfection, the route of administration, pharmacological considerationssuch as the activity, efficacy, pharmacokinetic, and toxicology profilesof the particular compounds employed, and whether a drug delivery systemis utilized. Administration of the drug combinations disclosed hereinshould generally be continued over a period of several weeks to severalmonths or years until virus titers reach acceptable levels, indicatingthat infection has been controlled or eradicated. Patients undergoingtreatment with the drug combinations disclosed herein can be routinelymonitored by measuring hepatitis viral RNA in patients' serum byslot-blot, dot-blot, or RT-PCR techniques, or by measurement ofhepatitis C viral antigens, such as surface antigens, in serum todetermine the effectiveness of therapy. Continuous analysis of the dataobtained by these methods permits modification of the treatment regimenduring therapy so that optimal amounts of each component in thecombination are administered, and so that the duration of treatment canbe determined as well. Thus, the treatment regimen/dosing schedule canbe rationally modified over the course of therapy so that the lowestamounts of each of the antiviral compounds used in combination whichtogether exhibit satisfactory anti-hepatitis C virus effectiveness areadministered, and so that administration of such antiviral compounds incombination is continued only so long as is necessary to successfullytreat the infection.

The present invention encompasses the use of the HCV serine proteaseinhibitors disclosed herein in various combinations with the foregoingand similar types of compounds having anti-HCV activity to treat orprevent HCV infections in patients, particularly those patients thathave HCV infections that have developed resistance to treatment byVX-950 and other standard protease inhibitors. For example, one or moreHCV serine protease inhibitors can be used in combination with: one ormore interferons or interferon derivatives having anti-HCV activity; oneor more non-interferon compounds having anti-HCV activity; or one ormore interferons or interferon derivatives having anti-HCV activity andone or more non-interferon compounds having anti-HCV activity. When usedin combination to treat or prevent HCV infection in a human patient, anyof the presently disclosed HCV serine protease inhibitors and foregoingcompounds having anti-HCV activity can be present in a pharmaceuticallyor anti-HCV effective amount. By virtue of their additive or synergisticeffects, when used in the combinations described above, each can also bepresent in a subclinical pharmaceutically effective or anti-HCVeffective amount, i.e., an amount that, if used alone, provides reducedpharmaceutical effectiveness in completely inhibiting or reducing theaccumulation of HCV virions and/or reducing or ameliorating conditionsor symptoms associated with HCV infection or pathogenesis in patientscompared to such HCV serine protease inhibitors and compounds havinganti-HCV activity when used in pharmaceutically effective amounts. Inaddition, the present invention encompasses the use of combinations ofHCV serine protease inhibitors and compounds having anti-HCV activity asdescribed above to treat or prevent HCV infections, where one or more ofthese inhibitors or compounds is present in a pharmaceutically effectiveamount, and the other(s) is(are) present in a subclinicalpharmaceutically-effective or anti-HCV effective amount(s) owing totheir additive or synergistic effects. As used herein, the term“additive effect” describes the combined effect of two (or more)pharmaceutically active agents that is equal to the sum of the effect ofeach agent given alone. A “synergistic effect” is one in which thecombined effect of two (or more) pharmaceutically active agents isgreater than the sum of the effect of each agent given alone.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease.Patients may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

A specific dosage and treatment regimen for any particular patient willdepend upon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, rate of excretion, drug combination, and the judgmentof the treating physician and the severity of the particular diseasebeing treated. The amount of active ingredients will also depend uponthe particular described compound and the presence or absence and thenature of the additional anti-viral agent in the composition.

Accordingly, the agents of the present application useful fortherapeutic treatment can be administered alone, in a composition with asuitable pharmaceutical carrier, or in combination with othertherapeutic agents. An effective amount of the agents to be administeredcan be determined on a case-by-case basis. Factors to be consideredusually include age, body weight, stage of the condition, other diseaseconditions, duration of the treatment, and the response to the initialtreatment. Typically, the agents are prepared as an injectable, eitheras a liquid solution or suspension. However, solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection canalso be prepared. The agent can also be formulated into anenteric-coated tablet or gel capsule according to known methods in theart. The agents of the present application may be administered in anyway which is medically acceptable which may depend on the identity ofthe agent and/or on the disease condition or injury being treated.Possible administration routes include injections, by parenteral routessuch as intravascular, intravenous, intraepidural or others, as well asoral, nasal, ophthalmic, rectal, topical, or pulmonary, e.g., byinhalation, aerosolization or nebulization. The agents may also bedirectly applied to tissue surfaces, e.g., during surgery. Sustainedrelease administration is also specifically included in the application,by such means as depot injections, transdermal patches, or erodibleimplants.

According to another embodiment, the invention provides a method fortreating a patient infected with or preventing infection by a viruscharacterized by a virally encoded serine protease that is necessary forthe life cycle of the virus by administering to said patient apharmaceutically acceptable composition of this invention. Preferably,the methods of this invention are used to treat a patient suffering froma HCV infection. Such treatment may completely eradicate the viralinfection or reduce the severity thereof. More preferably, the patientis a human being.

The term “treating” includes prophylactic (e.g., preventing) and/ortherapeutic treatments. The term “prophylactic or therapeutic” treatmentis art-recognized and includes administration to the host of one or moreof the subject compositions. If it is administered prior to clinicalmanifestation of the unwanted condition (e.g., disease or other unwantedstate of the host animal) then the treatment is prophylactic, (i.e., itprotects the host against developing the unwanted condition), whereas ifit is administered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

In an alternate embodiment, the methods of this invention additionallycomprise the step of administering to said patient an anti-viral agentpreferably an anti-HCV agent. Such anti-viral agents include, but arenot limited to, immunomodulatory agents, such as alpha-, beta-, andgamma-interferons, pegylated derivatized interferon-alpha compounds, andthymosin; other anti-viral agents, such as ribavirin and amantadine;other inhibitors of hepatitis C proteases (NS2-NS3 inhibitors andNS3-NS4A inhibitors); inhibitors of other targets in the HCV life cycle,including helicase and polymerase inhibitors; inhibitors of internalribosome entry; broad-spectrum viral inhibitors, such as IMPDHinhibitors (e.g., VX-497 and other IMPDH inhibitors disclosed in U.S.Pat. No. 5,807,876, mycophenolic acid and derivatives thereof); orcombinations of any of the above.

Such additional agent may be administered to said patient as part of asingle dosage form comprising both a compound of this invention and anadditional anti-viral agent. Alternatively the additional agent may beadministered separately from the compound of this invention, as part ofa multiple dosage form, wherein said additional agent is administeredprior to, together with or following a composition comprising a compoundof this invention.

In yet another embodiment the present invention provides a method ofpre-treating a biological substance intended for administration to apatient comprising the step of contacting said biological substance witha pharmaceutically acceptable composition comprising a compound of thisinvention. Such biological substances include, but are not limited to,blood and components thereof such as plasma, platelets, subpopulationsof blood cells and the like; organs such as kidney, liver, heart, lung,etc; sperm and ova; bone marrow and components thereof, and other fluidsto be infused into a patient such as saline, dextrose, etc.

According to another embodiment the invention provides methods oftreating materials that may potentially come into contact with a viruscharacterized by a virally encoded serine protease necessary for itslife cycle. This method comprises the step of contacting said materialwith a compound according to the invention. Such materials include, butare not limited to, surgical instruments and garments; laboratoryinstruments and garments; blood collection apparatuses and materials;and invasive devices, such as shunts, stents, etc.

In another embodiment, the compounds of this invention may be used aslaboratory tools to aid in the isolation of a virally encoded serineprotease. This method comprises the steps of providing a compound ofthis invention attached to a solid support; contacting said solidsupport with a sample containing a viral serine protease underconditions that cause said protease to bind to said solid support; andeluting said serine protease from said solid support. Preferably, theviral serine protease isolated by this method is HCV NS3 protease. Moreparticularly, it is a mutant HCV NS3 protease that is resistant totreatment by VX-905 and/or BILN 2061 as described herein. Exemplary suchproteases includes those described herein as having mutant (i.e.,non-wild-type) residues at positions 36, 41, 43, 54, 148, 155, and/or156 of a protein of SEQ ID NO:2.

EXEMPLIFICATION

The disclosure now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present disclosure, and are not intended to limit the disclosure.

Example 1 Patient Population and Study Design

Thirty four patients infected with genotype 1 HCV who were enrolled in aphase 1b randomized, blinded, dose-escalation clinical trial for VX-950(Study VX04-950-101) were subjects of the study. All patients werebetween 18 and 65 years of age, had baseline HCV RNA levels of at least10⁵ IU/mL, and were hepatitis B virus (HBV) and HIV negative. Patientswere divided into 3 groups receiving 450 (q8h), 750 (q8h), or 1250(q12h) mg VX-950 for 14 consecutive days, with 2 placebo patients ineach dosing group. Four milliliter (mL) blood samples were collectedfrom study patients at 3 time points: the day before dosing (baselinesamples), at day 14 of dosing or end of treatment (ETR sample), and 7 to10 days after the last dose of study drug (follow-up sample). Blood wascollected by venipuncture of a forearm vein into tubes containing EDTA(K₂) anticoagulant. Plasma was separated by 10 min centrifugation,frozen, and stored at −80° C. for less than 6 months. Virions wereisolated from this plasma for sequence analysis.

Example 2 Amplification and Sequencing of the HCV NS3 Protease fromPatient Plasma

Sequence analysis of HCV was done by semi-nested reverse-transcriptasepolymerase chain reaction (RT-PCR) amplification of a HCV RNA fragmentcontaining the full 534 base pair (bp) NS3 serine protease region fromplasma virus. The virions were lysed under denaturing conditions, andthe HCV RNA was isolated using a standard commercial silica-gelmembrane-binding method (QIAamp Viral RNA Minikit; Qiagen, Valencia,Calif.). A complementary DNA (cDNA) fragment containing the NS3 serineprotease region was synthesized from viral RNA and amplified using acommercial 1-step reverse transcriptase PCR (Superscript III RNaseH-Reverse Transcriptase with High Fidelity Platinum Taq DNA Polymerase;Invitrogen Corp, Carlsbad, Calif.). A 912 bp coding region of NS3 wasamplified using primers flanking the NS3 region (NS3-1b-1s:GGCGTGTGGGGACATCATC; and NS3-1b-3a: GGTGGAGTACGTGATGGGGC). Two rounds ofnest PCR were performed for each sample at a final concentration of 0.5μM primer (Invitrogen Custom Primers), 0.2 mM dNTPs (Invitrogen Corp),1.2 mM MgSO₄, and 34.8 units of RNA guard (Porcine RNase Inhibitor,Amersham Biosciences) in 1× proprietary reaction buffer. Reactionmixtures were initially incubated for a 30 min reverse transcriptionreaction at 47° C. followed by a 3 min denaturation step at 94° C. andthen 30 cycles of 94° C. for 30 sec, 51° C. for 30 sec, and 68° C. for45 sec. The first PCR product was diluted 1:10 and used in anothersemi-nested reaction using 1.25 units of AccuPrime Pfx DNA polymerase,0.5 μM primer (NS3-1b-1s and NS3-1b-4a; CATATACGCTCCAAAGCCCA), 0.3 mMdNTPs, 1 mM MgSO₄, and 1× reaction buffer. The DNA products from theouter PCR were denatured for 3 min at 94° C. and amplified with 30cycles of 94° C. for 30s, 53° C. for 30s, and 68° C. for 30s. The DNAfrom this PCR was then separated on a 1% agarose gel, and theappropriately sized product (830 bp) was purified using the QIAquick GelExtraction Kit (Qiagen). Isolated DNA was then cloned using the ZeroBlunt TOPO PCR Cloning Kit (Invitrogen Corp). Cloning plates were sentto SeqWright (Houston, Tex.) where 96 clones were amplified andsequenced per patient per time point.

Example 3 Sequence Alignment and Phylogenetic Analysis

Sequences were aligned and analyzed for mutations using the softwareMutational Surveyor (SoftGenetics, State College, Pa.). The N-terminal543 nucleotides (181 amino acids) of NS3 protease were analyzed. Aconsensus sequence for each patient was developed from an average of 84baseline sequences, and an average of 81 sequences were obtained foreach patient at Day 14 and at follow-up (7 to 10 days after the lastdose of study drug). Phylogenetic trees were made using PHYLIP(Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package) version3.5c. Distributed by the author. Department of Genetics, University ofWashington, Seattle, Wash.) Dnadist and Quick Tree(http://www.hcv.lanl.gov/content/hcv-db, accessed June 2005).

Example 4 Expression and Purification of Recombinant NS3 ProteaseProteins

A DNA fragment encoding Met¹-Ser¹⁸¹ of the HCV NS3 protease wasamplified from selected plasmid clones of patient isolates usingoligonucleotides specific for each HCV variant. The DNA fragment wascloned into the Escherichia coli expression plasmid pBEV11 leading to a181-residue HCV NS3 protease domain followed by a C-terminalhexa-histidine tag. Recombinant 6X-His-tagged NS3 proteases were thenexpressed in E. coli using a leaky expression method as previouslypublished (4). Five to seven isolated colonies of E. coli BL21 (DE3)freshly transformed with the NS3 protease expression plasmids were usedto inoculate a 5 mL LB medium with 100 μg/mL carbenicillin. These seedcultures were incubated at 37° C. with shaking (250 rpm) until reachingan OD₆₂₀ between 0.3 and 1, then used to inoculate 50 mL 4×TY broth (32g/L tryptone, 20 g/L, yeast extract, 5 g/L NaCl) containing 100carbenicillin in 250 mL Erlenmeyer flasks at an initial OD₆₂₀ of ˜0.010.The expression cultures were incubated for 24 hours at ambienttemperature (˜25° C.) with shaking at 250 rpm. The cells were harvestedby centrifugation at 3000×g for 30 min, the pellets were rapidly frozenin an −80° C. ethanol bath and stored at −80° C. until the protease waspurified.

Recombinant proteases were purified from E. coli using a modification ofa published method (7). Frozen cell pellets were thawed and re-suspendedin 6.8 mL of cold Buffer A (50 mM N-2-Hydroxyethylpiperizine-N′-ethanesulfonic acid [HEPES, pH 8.0]; 1 M NaCl; 10%[vol/vol] glycerol; 5 mM imidazole; 5 mM β-mercaptoethanol; 0.1% Octylβ-D-glucopyranoside [Sigma, Saint Louis, Mo.]; 2 μg/mL Leupeptin [Sigma,Saint Louis, Mo.]; 1 μg/mL E-64 [Sigma, Saint Louis, Mo.]; 2 μg/mLPepstatin A [Sigma, Saint Louis, Mo.]). The cells were lysed by theaddition of 0.8 mL 10× BugBuster reagent (Novogen/EMD Biosciences,Madison, Wis.) and 8 μL of 1000× Benzonuclease (Novogen/EMD Biosciences,Madison, Wis.) followed by gentle rocking at 4° C. for 30 min. Celllysates were centrifuged at 16,000×g to remove insoluble material. Eachsupernatant was applied to a 0.25 mL bed volume of TALON metal affinityresin (BD Biosciences, Palo Alto, Calif.) equilibrated to Buffer A indisposable polypropylene columns (Biorad, Hercules, Calif.). Thelysate/resin slurries were rocked at 4° C. for 30 min. The lysates weredrained from the column and the resin washed with 3-5 mL volumes ofBuffer A. Two aliquots (0.25 mL each) of Buffer B (50 mM HEPES [pH 8.0];1 M NaCl; 25% [vol/vol] glycerol; 300 mM imidazole; 5 mMβ-mercaptoethanol; 0.1% Octyl β-D-glucopyranoside; 2 μg/mL Leupeptin; 1μg/mL E-64; 2 μg/mL Pepstatin A]) were used to elute protein bound tothe column, the two fractions were pooled, divided into small aliquotsand stored at −80° C. The concentration of the eluted protein wasdetermined using a Coomassie protein assay (Biorad, Hercules, Calif.)according to the manufacturers instructions with a bovine serum albuminstandard. Purity of the protease was estimated using 1D Image AnalysisSoftware (Kodak, Rochester, N.Y.) from protein samples resolved ondenaturing acrylamide gels (SDS-PAGE) stained with Biosafe CoomassieBlue (Biorad, Hercules, Calif.).

Example 5 Enzymatic Assay for the HCV NS3 Serine Protease Domain

In vitro protease activity was assayed as published (7) in 96-wellmicrotiter plates (Corning NBS 3990) with both VX-950 and BILN 2061protease inhibitors. BILN 2061 is a HCV NS3.4A protease inhibitordiscovered by Boehringher Ingelheim, Laval, Quebec, Canada. Briefly,protease was incubated with 5 μM co-factor KK4A at 25° C. for 10 min andat 30° C. for 10 min. Protease inhibitor (VX-950 or BILN 2061), seriallydiluted in DMSO, was added and incubated for an additional 15 min at 30°C. The reaction was initiated by the addition of 5 μM RET-S1 (AnaspecInc. San Jose, Calif.), an internally quenched fluorogenic depsipeptidesubstrate, and incubated at 30° C. Product release was monitored for 20min (excitation at 360 nm and emission at 500 nm) in a TecanSpectraFluor Plus plate reader. Data were fitted with a simple IC₅₀equation: Y=V₀/(1+(X/IC₅₀)).

Example 6 K_(m) Determination of HCV NS3 Serine Protease Domain Proteins

Substrate kinetic parameters were determined with an internally quenchedfluorogenic depsipeptide substrate, RET-S1 (Ac-DED(EDANS)EEαAbuψ[COO]ASK(DABCYL)-NH2) (Taliani et al., (1996) Anal. Biochem. 240 (1), 60-67).Protease was pre-incubated with 5 μM co-factor peptide KK4A(KKGSVVIVGRIVLSGK) (Landro et al., (1997) Biochemistry 36 (31),9340-9348) in 50 mM HEPES (pH 7.8), 100 mM NaCl, 20% glycerol, 5 mMdithiothreitol at 25° C. for 10 min and at 30° C. for 10 min. Thereaction was initiated by the addition of the RET-S1 substrate (AnaspecIncorporated, San Jose, Calif.) and incubated for 10 min at 30° C. Totalassay volume was 100 μL. The reaction was quenched by the addition of 25μL 10% trifluoroacetic acid (TFA). Reaction products were separated on areverse phase microbore high performance liquid chromatography column(Penomenex Jupiter 5μ C18 300 A column, 150×2.0 mm), which was heated to40° C. The flow rate was 0.2 ml/min, with H2O/0.1% TFA (solvent A) andacetonitrile/0.1% TFA (solvent B). A linear gradient was used asfollows: 5% to 30% solvent B over 1 min, 30% to 40% solvent B over 15min, then 40% to 100% solvent B over 1 min, 3 min isocratic, followed by100% to 5% B in 1 min, and equilibration at 5% B for 10 min. TheDABCYL-peptide product was detected at 500 nm and typically elutedaround 17 min. K_(m) was determined by fitting the data to theMichaelis-Menten equation with GraphPrism software.

Example 7 Determination of Telaprevir (VX-950) K_(i(app,1h)) of the HCVNS3 Serine Protease Domain Variants

Sensitivity of the NS3 protease domain variants to telaprevir wasdetermined in 96-well microtiter plates (Corning NBS 3990; Corning,N.Y.) as published previously (Lin et al., (2004) J. Biol. Chem. 279(17), 17508-17514). Briefly, the NS3 protease domain was pre-incubatedwith 5 μM KK4A peptide in 50 mM HEPES (pH 7.8), 100 mM NaCl, 20%glycerol, 5 mM dithiothreitol at 25° C. for 10 min and at 30° C. for 10min. Telaprevir, serially diluted in DMSO, was added to the proteasemixture and incubated for an additional 60 min at 30° C. The reactionwas started by the addition of 5 μM RET-S1 substrate and incubated at30° C. Product release was monitored for 20 min (excitation at 360 nmand emission at 500 nm) in a Tecan SpectraFluor Plus plate reader (TecanUS, Durham, N.C.). Total assay volume was 100 μl. Protease concentrationwas chosen such that 10-20% of the substrate was turned over during thecourse of the assay. To calculate apparent inhibition constant(K_(i(app,1h))) values, data were fit to the integrated form ofMorrison's equation for tight binding inhibition (38) using theGraphPrism software. Steady-state assays showed that the wild typeenzyme and all R155K/T/I/S variants had a K_(m) for RET-S1 that washigher than the limit of detection (100 μM). Thus, the K_(m) was set to100 μM for calculating K_(i(app,1h)) values. Inhibitor studies werecarried out at a substrate concentration (5 M) that is significantlybelow the K_(m). Therefore, the deviation between the true K_(m) and theK_(m) used in calculations should have a negligible effect in thecalculation of K_(i(app,1h)).

Example 8 Sequence Analysis of Baseline Samples

The consensus sequence for each patient's HCV population was derivedfrom an average of 84 independent plasmid clones containing HCV cDNA.Phylogenetic analysis of the consensus sequences indicated thatsequences were patient specific (FIG. 1). The average intra-patientamino acid quasispecies complexity (Shannon entropy) and diversity(Hamming distance) were low (0.332±0.109 and 0.421±0.195, respectively),and no correlation of quasispecies heterogeneity with HCV RNA plasmaconcentration at baseline was observed. The inter-patient amino aciddiversity (individual consensus compared to genotype 1a or 1b consensussequence of the patients in this trial) was 1.3% for genotype 1a and 2%for genotype 1b. Structural modeling analysis predicted that amino aciddifferences observed between consensus sequences of all patients withina subtype would have little or no impact on VX-950 binding.

Patient-specific protease clones were then expressed and tested forinhibition by VX-950. In agreement with the modeling observation, therewere no significant differences in the enzymatic IC₅₀ values of theseproteases derived from different patient isolates within a specificsubtype. However, the average IC₅₀ for genotype 1b patients was slightlyhigher than for genotype 1a patients (FIG. 2). This finding isconsistent with previous in vitro results measuring the K_(i) value forHCV-H (1a) and HCV Con1 (1b) (7). Modeling analysis of 1a versus 1bgenotypes suggested that the key difference that may affect theinhibitor/substrate binding is at the residue position 132, whereasother differences are located outside the binding pocket. The Val¹³²side-chain of the genotype 1b protease makes only one van der Waalscontact with the P3 terbutyl-glycine group of VX-950, while the Ile¹³²side-chain of the genotype 1a protease makes 2 contacts. This structuraldifference in interactions is consistent with the experimental data thatshows a lower enzyme IC₅₀ of VX-950 with the genotype 1a proteasescompared to the genotype 1b proteases. Although there is a slightdifference between subtypes, both subtypes are still clearly sensitiveto VX-950. In conclusion, despite the observed sequence diversity in theHCV NS3 serine protease, genotype 1 patients are expected to beresponsive to treatment with the protease inhibitor VX-950. The clinicaldata supports this finding as no significant difference in viralresponse to VX-950 was observed.

Example 9 Genotypic Data: Sequence Analysis of ETR and Follow-Up Samples

The HCV NS3 protease sequences at end of treatment (ETR) were comparedto the consensus sequences at baseline for each patient to identifypotential resistance mutations. An average of 80 sequences was obtainedfor each ETR sample, and the percent of variants at each of 181positions was calculated. Initially, an increase in frequency of 5% orgreater at any single amino acid position of the ETR sample compared tothe baseline was considered to be a potential resistance mutation. The5% cut off value was used because this was the lower level ofsensitivity of our sequencing protocol, based on the number of clonesanalyzed and the error rate of the PCR. Changes at sites that werepolymorphic at baseline were not considered resistance mutations.Changes at sites which were only present at end of dosing and that wereobserved in multiple patients were considered potential resistancemutations and these were then analyzed in all the patients.

For analysis, patients were split into groups based on viral load(plasma HCV RNA levels) response to VX-950. Patients were grouped into“initial responders” or “continued responders” in the viral dynamicanalysis. In viral sequence analysis, the “initial responder” group wasfurther divided into two groups based on the increase in plasma HCV RNAafter the initial decline. Patients who had less than a 0.75 log₁₀increase from the lowest measured HCV RNA level to end of dosing (Day14) were categorized as patients with an HCV RNA “plateau”. Those whohad greater than a 0.75 log₁₀ increase in plasma HCV RNA werecategorized as patients with HCV RNA “rebound”. Normal fluctuation inHCV RNA in an untreated patient is about 0.5 log₁₀, and these groupingswere based on antiviral response as well as viral mutational pattern.There were 2 patients for whom these categories were inconsistentbetween the 2 types of analyses. Patient 12308 had an increase of 0.05log₁₀ from nadir to end of dosing (Day 14) and was categorized as“plateau” for the sequence analysis, while for the viral dynamicanalysis 12308 was placed in the “continued responders” group. Patient3112 had undetectable plasma HCV RNA (<10 IU/ml) at Day 11, butdetectable plasma HCV RNA of 35 IU/ml at end of dosing (Day 14). Thisincrease in HCV RNA caused the patient to be placed in “initialresponders” group for viral dynamic analysis; however, in the sequenceanalysis the level of HCV RNA remained undetectable by the sequencingassay and the patient was therefore grouped into the “continuedresponders” group.

Sequence analysis grouped patients by: no response (placebo); declinefollowed by rebound during dosing (rebound); decline followed by plateauduring dosing (plateau); and continued decline throughout dosing(continued responders) (FIG. 3). Additionally, within these groups,patients were analyzed by dose group and genotype subgroup (1a or 1b).Lastly, mutations were analyzed in follow-up samples collected 7 to 10days after withdrawal of VX-950 to monitor the persistence of anymutations as well as any shift from baseline variants. An average of 81clones were analyzed for each follow-up sample. Complete sequenceanalysis is available for 28 of the patients, and analysis of theremaining 6 patients is currently in progress. Analysis is ongoing for 2patients with rebound, 3 with plateau, and 1 with continued decline. Inthe first group of placebo patients (n=6), there were no significantchanges from baseline at any position in any patient.

Example 10 Patients with HCV RNA Rebound During Dosing

There were 13 patients who initially responded to VX-950 with a greaterthan 2 log₁₀ drop in HCV RNA, but eventually began to rebound whilestill being dosed with VX-950. Of these 13 patients, 6 were in the 450mg q8h dose group, 1 was in the 750 mg q8h dose group, and 6 were in the1250 mg q12h dose group. Complete sequence analysis is available for 11of these patients at Day 14 (Table 1). All of these patients had asignificant increase in mutations at position 36. At position 36, thewild-type valine was mutated to an alanine (V36A) in genotype 1bpatients (mean 60%, range 31%-86%) and to either an alanine or amethionine (V36M) in genotype 1a patients (mean 62%, range 18%-90%). Theabsence of V36M in subtype 1b is likely due to the requirement of 2nucleotide substitutions in subtype 1b versus only a single change insubtype 1a. The V36A mutation requires a single nucleotide change ineither subtype 1a or 1b. Mutation to glycine at position 36 (V36G) wasalso seen in genotype 1b patients and to a leucine (V36L) in 1apatients, but at a much lower frequency. Three patients also had amutation at position 54 from a threonine to an alanine (T54A) (mean 35%,range 8%-67%) and less frequently to a serine (T54S). Interestingly, themutations at positions 36 and 54 appear to be mutually exclusive.Additionally, all patients in this group who were genotype 1a containeda mutation at position 155 from an arginine to either lysine (R155K) orthreonine (R155T) (mean 60%, range 22%-99%), and less frequently toisoleucine (R155I), serine (R155S), methionine (R155M), or glycine(R155G). The observation that these mutations at R155 is restricted tosubtype 1a patients is again likely due to the requirement for 2nucleotide changes from baseline in the 1b patients versus a singlenucleotide change in 1a patients. In follow-up samples from this group,wild-type virus began to re-emerge, but all mutations seen at ETR werestill present, although at different frequencies potentially due todifferences in viral fitness. No sequencing samples are available forthe time point at which rebound was first observed for each patient, soit is not clear if other mutations were present earlier.

Example 11 Patients with Plateaued HCV RNA Response During Dosing

In the next group (n=8), patients responded to VX-950 initially, buttheir HCV RNA response stabilized and did not continue to decline,although no increase in HCV RNA was seen. Two of these patients were inthe 450 mg q8h dose group, 2 were in the 750 mg q8h dose group, and 4were in the 1250 mg q12h dose group. Analysis is complete for 5 of thesepatients at Day 14 (Table 1). All patients developed a mutation atposition 156 from an alanine to either valine (A156V) or threonine(A156T), and very infrequently to a serine (A156S) or isoleucine(A156I). The A156V/T mutation was seen at a very high frequency in 3 ofthe patients in this group (100%), and no other mutation was seen inthese patients. The other 2 patients (02104 and 12308) only had 8% A156Tand 30% A156V mutant virus, respectively, but these patients also hadmutations at other positions (47% V36A/M and 36% R155K/T for patient02104; 68% T54A for patient 12308). One patient from the rebound group,02102, also had a similar frequency of mutations (11%) at position 156.At follow-up time points, the mutation at position 156 was almostcompletely replaced by either wild-type virus or virus with mutations atpositions 36, 54, and/or 155. A change to serine at position 156 (A156S)was the dominant mutation seen during in vitro resistance studies ofVX-950 (7). The A156V/T mutations were also identified ascross-resistant to both VX-950 and BILN 2061 in an in vitro study (7).

Example 12 Patients with Continued Response During Dosing

The remaining 7 patients responded to VX-950 with a continuous declinein HCV RNA levels throughout the 14-day dosing period. Five of thesepatients were in the 750 mg q8h dose group and 2 were in the 450 mg q8hdose group. These patients had very low levels of HCV RNA at Day 14 (64IU/mL to undetectable (<10 IU/mL)), and sequencing data could not beobtained at this time point. However, HCV cDNA was successfullyamplified from the follow-up samples for these patients, and analysis iscomplete for 6 of these 7 patients (Table 1). The genotype 1a patient(03205) who had reached undetectable levels by Day 14 had 3 mutations,V36A/M (67%), T54A (11%), and R155K/T (26%), at the follow-up timepoint. Two genotype 1b patients had the V36A (21% and 25%) and T54Amutations (54% and 20%), and another two 1b patients had only low levelsof the V36A (3% and 9%) and the T54A (6% and 1%) mutations at follow up.In the last 1b patient who had undetectable HCV RNA levels at Day 14(<10 IU/mL), no mutations were detected at follow up.

Example 13 Frequency of Double Mutations

Frequency of double mutations that were found in patients were alsoanalyzed. The mutations at position 36 were found in combination withmutations at both positions 155 (36/155 double mutation) and 156 (36/156double mutation). Only genotype 1a patients had the 36/155 doublemutation, which was seen in all eight 1a patients analyzed in therebound group (mean 27.6%, range 10%-77%) and in one of two 1a patientsin the plateau group (9%) at Day 14 ETR. The 36/156 double mutation wasmuch less frequent, and found in 3 patients in the rebound group (3%,range 1%-5%) and in 2 patients in the plateau group (1% and 4%) at Day14 ETR. Double mutations were also found in the follow-up samples at asimilar frequency for 36/155 but at a lower frequency for 36/156 ascompared with the ETR samples. For the group of patients that continuedto decline, only 1 patient had the 36/155 double mutation, which waspresent at 5%.

The frequency of mutations found either alone or in combination is shownin FIG. 4. A summary of the resistance patterns is shown in FIG. 5,which depicts the average percent of mutated amino acids for eachpatient group.

Example 14 Phenotypic Data: Enzymatic IC₅₀ Analysis of ResistanceMutations

Since an average of 82 independent sequencing clones were subjected togenotypic analysis, there was a mixture of virions in each patient, asshown in Table 1. The enzyme IC₅₀ of all mutants seen in the abovesequence analysis (V36A/M/G, T54A/S, R155K/T/M/G/S, and A156TN/S) aswell as any observed combinations of these mutations found in vivo weredetermined in at least 2 different patient-specific genetic backgrounds.The baseline IC₅₀ for all patients within a genotype were similar, asreported in Example 6. Enzyme IC₅₀ values of resistant proteases arereported as fold change compared to the genotype 1a HCV-H referencestrain. The IC₅₀ of this reference strain was found to be 64 nM in thisenzyme assay.

FIG. 5 shows the enzyme IC₅₀ values and the fold change over thereference strain for single mutants. The values that are similar for anyamino acid change at a given position are grouped together in boxes inthis figure. A single mutation at position 36 confers low levels ofresistance to VX-950. The V36A/M/L mutations show about a 1.5- to10-fold increase in enzyme IC₅₀, regardless of the specific amino acidchange. The substitution at position 54 from a threonine to a serine(T54S) does not significantly increase IC₅₀. However the T54A mutationgives about a 10-fold increase in enzyme IC₅₀. The substitution atposition 155 also confers fairly low levels of resistance (about a 5- to15-fold increase in IC₅₀), for any of the changes from an arginine to athreonine (R155T), lysine (R155K), methionine (R155M), or serine(R155S). The mutation at position 156 from an alanine to a valine(A156V), threonine (A156T), or isoleucine (A156I) confers high levels ofresistance (about a 400- to 500-fold increase in enzyme IC₅₀).Interestingly, the A156S mutation is much less resistant to VX-950 (onlya 22-fold increase in IC₅₀) than the other amino acid changes at thisposition, which is consistent with the in vitro resistance studies (6,7).

FIG. 6 shows the actual enzyme IC₅₀ values as well as the fold changeover the reference strain for double mutants. Double mutations at thesepositions give higher levels of resistance than any single mutation. Amutation at position 36 combined with either a mutation at position 155or 156 gives about an additional 10-fold increase in resistance over therespective single mutants. Table 2 lists the actual IC₅₀ values for allmutants tested against both VX-950 as well as another HCV proteaseinhibitor, BILN 2061. The mutations at positions 36 and 54 affectsusceptibility to VX-950 much more than BILN 2061, whereas the mutationat residue 155 confers much higher levels of resistance to BILN 2061.The mutations at position 156 as well as the double mutations all conferhigh levels of resistance against both inhibitors. However, the A156Smutant is more resistant to VX-950, which has been shown previously invitro (7). Table 3 shows the mean and standard deviation values of theenzyme IC₅₀ and fold change over the reference strain of all mutationsat a given position.

Example 15 Average Phenotypes for Each Patient

The genotypic analyses performed here allowed a detailed examination ofthe relative proportion of different resistant mutants within eachpatient. To better understand the level of phenotypic resistanceconferred by these viral mixtures, exploratory analyses were done toobtain an average phenotype for each patient. The percent of virionswith a given single or double mutation was multiplied by the averagefold change in enzyme IC₅₀ for that given mutant (Table 3), and then allvalues were added for each patient. The relative number (value dividedby 100) is shown in the last column of Table 1. The mean value for therebound group of patients was 27 at Day 14 (range 6-70) and 15 atfollow-up (range 3-29). The mean value for the plateau group of patientswas 272 at Day 14 (range 26-466) and 32 at follow-up (range 1-126). Thelast group of patients that continued to decline had a mean value of 4at follow-up (range 0-10). From these very preliminary calculations, itseems that the level of resistance in all groups of patients declinesafter the last dose (from Day 14 to follow-up), which is not unexpected,as the drug pressure is no longer present for selection of resistantvirions. It also appears that the rebound group of patients has lowerlevels of resistance than the patients that plateaued. An analysiscorrelating pharmacokinetic data with resistance patterns is currentlyunderway.

Example 16 Fitness Analysis of Resistance Mutations

Another important aspect of these viral variants in addition to theirresistance profile is the level of fitness. The viral fitness of singleand double mutants was calculated (Table 4). The absolute number ofinfectious units (IU/mL) of each HCV variant in an individual patient ata given time point was determined. For example, the absolute number ofinfectious units of variants with the A156V/T mutation in Patient 03201at Day 14 was calculated as:VL_(d14 (A156V/T, 3201))=VL_(d14 (whole, 3201))×% A156V/T_(d14 (3201)).Total HCV RNA at either end-of-dosing (Day 14, VL_(d14 (whole, 3201)))or follow-up (Day 21, VL_(d21 (whole, 3201))) was taken from theanalysis of plasma HCV RNA levels, as determined by the Roche COBASTaqman HCV assay. Undetectable levels of HCV RNA were considered to be 5IU/mL. The percentage of each individual variant group (e.g., A156V/T)at either end-of-dosing (Day 14, % A156V/T_(d14 (3201))) or follow-up(Day 21, % A156V/T_(d21 (3201))) was taken from the sequence analysisdescribed above (Table 1). Next, the fold of net increase in plasma HCVviral load (NVL) for each HCV variant and the whole population of HCV inthe plasma in the individual patient after dosing (Day 14 to Day 21) wasdetermined. For example:

NVL _((A156V/T,3201)) =VL _(d21(A156V/T,3201)) /VL _(d14(A156V/T,3201))

NVL _((whole,3201)) =VL _(d21(whole,3201)) /VL _(d14(whole,3201))

The NVL of each HCV variant was normalized to that of the whole plasmaHCV population in each patient: %NVL_((A156V/T, 3201))=NVL_((A156V/T, 3201))/NVL_((whole, 3201)).

The normalized fold of net increase in viral load of each HCV variant inindividual patients were then converted into log₁₀-converted values:Log₁₀[% NVL_((A156V/T, 3201))], and the average value for a variant wasdetermined from all patients. The fitness of the HCV mutant was thencalculated: Fitness for A156V/T mutation=10_(Log(fitness, A156V/T)).This relative fitness score was analyzed with the resistance level ofeach mutant. As shown in FIG. 7, all mutants were less fit thanwild-type and, in general, using this analysis there is an inversecorrelation between fitness and resistance of single mutants. The doublemutant 36/155, however, has a significant increase in resistance withless impact on fitness. This may be due to interaction of the mutationscompensating for the loss of fitness, while still conferring resistance.

Example 17 X-Ray Structure of the R155K Variant Protease

Purified R155K variant of the HCV-H strain protease domain, in a complexwith an NS4A peptide co-factor (Kim et al., (1996) Cell 87 (2),343-355), at a concentration of 8.0 mg/ml was used for crystallizationtrials. The protein crystals were grown over a reservoir liquid of 0.1 MMES (pH 6.2), 1.4 M NaCl, 0.3 M KH₂PO₄, and 10 mM β-mercaptoethanol.Single crystals were obtained in the hanging droplets afterequilibrating over two days. A single crystal with dimensions of0.15×0.15×0.35 mm was transferred into cryo-protectant solution ofmother liquid with 25% glycerol added shortly prior to be flush-cooledto 100K in nitrogen gas stream. The diffraction images were collectedusing a CCD4 image plate instrument mounted on an ALS beam line 5,01.Data at 2.5-Å resolution was indexed and integrated using HKL (2000, HKLIncorporated, Charlottesville, Va.) and CCP4 software. The crystalsbelong to space group R32 with unit cell dimensions of a=225.31 Å,b=225.31 Å, c=75.66 Å, α=90.00°, β=90.00° and γ=120.00°. There are 5% ofdata assigned for testing free R-factor in the later refinements. Thecrystals of the R155K variant studied here have an identicalcrystallographic lattice to that of the wild-type protease NS3-4Apublished previously (Kim et al., supra). The published NS3-4A proteasedomain (PDB code: 1A1R) was used to perform the initial rigid-body andpositional refinement of the model. The difference in the side chains ofresidue 155 was confirmed to be a Lys instead of Arg in the electrondensity map. The protein molecule was visually inspected against theelectron density map using QUANTA programs. Further inclusion of solventmolecules in the refinement and individual B-factor refinement at aresolution range of 20.0 to 2.5 Å reduced the R-factor and free R-factorto 22.0% and 24.7%, respectively. Residues included in the refined modelrange from amino acids 1 to 181 of the NS3 protease domain and residues21 to 39 of the NS4A cofactor for crystallographic independent moleculeand two Zn metal ions.

Example 18 Computational Modeling

Telaprevir was modeled into the active site of the R155K variant NS3protease domain using the X-ray crystal structure of the R155K variantapo-enzyme in a complex with an NS4A cofactor peptide, following theprocedure described previously (Lin et al., supra). The ketoamide groupof telaprevir was modeled to form a covalent adduct with the Ser¹³⁹side-chain with a si-face attachment. This binding mode was observed foranalogous ketoamide inhibitors (Perth et al., (2004) Bioorg. Med. Chem.Lett. 14 (6), 1441-1446) and ketoacid inhibitors (Di Marco et al.,(2000) J. Biol. Chem. 275 (10), 7152-7157). The main-chain of theinhibitor was overlaid with the analogous main-chain of these ketoamideand ketoacid inhibitors such that the telaprevir main-chain makes allthe following backbone hydrogen bonds: P1 NH with Lys¹⁵⁵ carbonyl, P3carbonyl with Ala¹⁵⁷ NH, P3 NH with Ala¹⁵⁷ carbonyl, and P4 cap carbonylwith NH of Cys¹⁵⁹. In this binding mode, the P2 group of telaprevir wasplaced in the S2 pocket without any need to move the Lys¹⁵⁵ side-chain.The t-butyl and the cyclohexyl groups of telaprevir were placed in theS3 and S4 pockets, respectively. The inhibitor was energy minimized intwo stages. In the first stage, only the inhibitor and the side-chainatoms of Arg¹²³, Lys¹⁵⁵, and Asp¹⁶⁸ of the protease were allowed to moveduring energy minimization for 1000 steps. In the second stage, all theside-chain atoms of the protease were allowed to move along with theinhibitor for 1000 additional steps. This modeled structure closelymimics the telaprevir model in the active site of the wild-type NS3protease described previously (Lin et al., supra). No significant shiftsin the positions of Lys¹⁵⁵ side-chain or the other active residueside-chains were observed. The same procedure was repeated for dockingtelaprevir into the active site of the R155T variant of the NS3 proteasedomain. However, the enzyme structure in this case is not the X-raycrystal structure, but a model built using the R155K variant crystalstructure. The Lys¹⁵⁵ residue was replaced with Thr side-chain and wasminimized by holding all the atoms of the enzyme fixed except for theThr¹⁵⁵ side-chain. In this model, the hydroxyl group of the Thr¹⁵⁵side-chain forms a hydrogen bond with the side-chain of Asp⁸¹. Allmodeling and minimization procedures were carried out using the QUANTAmolecular modeling software (Accelrys Incorporated, San Diego, Calif.).

Example 19 Study Results

Results from enzymatic assays and structural studies are presented here,in addition to the discussions above for certain variants of theinvention.

1) Substitutions at Arg¹⁵⁵ of the NS3 Protease Confer Low-LevelResistance to Telaprevir in HCV Replicon Cells

To determine whether the observed substitutions of Arg¹⁵⁵ of the HCV NS3protease domain are sufficient to confer resistance to telaprevir(VX-950), several substitutions at NS3 residue 155 (R155K, R155T, R155S,R155I, R155M, or R155G) were introduced into a high-efficiencysubgenomic replicon plasmid (Con1-mADE). Stable HCV replicon cells weregenerated for each of these variants, indicating that replacement of NS3Arg¹⁵⁵ with a different residue did not abolish HCV RNA replication incells. The average 48-h IC₅₀ value of telaprevir in the wild-type HCVreplicon Con1-mADE cells was 0.485±0.108 μM, which is slightly higherthan what had been determined previously (0.354 μM) in Con1-based HCVreplicon cells with a different set of adaptive mutations (24-2)(25,41). Two major Arg¹⁵⁵ variants, R155K and R155T, which were observedin a phase 1b trial of telaprevir alone, had average 48-h telaprevirIC₅₀ values of 3.59±0.28 μM for R155K and 9.60±0.87 μM for R155T. Thiscorresponds to a 7.4- or 20-fold increase, respectively, compared to thewild-type Con1-mADE replicons (Table I). Similar decreases insensitivity to telaprevir were observed in HCV replicon cells containingthe other four minor variants at Arg¹⁵⁵: R155S, R155I, R155M, or R155G;Table I. These 4 variants were found at much lower frequency thanR155K/T in the telaprevir phase 1b trial. The replicon 48-h IC₅₀ valuesfor these four variants were 1.97±0.21 μM (R155S), 11.7±2.5 μM (R155I),2.68±0.21 μM (R155M), and 3.58±0.24 μM (R155G), which corresponds to a4.1-, 24-, 5.5-, and 7.4-fold, respectively, loss of sensitivity totelaprevir (Table I). These results indicate that substitutions of NS3Arg¹⁵⁵ led to low-level (<25-fold) resistance to telaprevir in HCVreplicon cells, independent of the physical properties of thesubstituted residue, which include a positive charged residue (Lys), ahydrophilic residue (Thr or Ser), a hydrophobic residue (Ile or Met), ora residue that lacks a side chain (Gly).

TABLE I Demonstration of Resistance in HCV Replicon Cell assays RepliconIC₅₀ of Variants telaprevir (μM) Fold change Wild-type 0.485 ± 0.108 1.0± 0.2 R155K 3.59 ± 0.28 7.4 ± 0.6 R155T 9.60 ± 0.87 20 ± 2  R155S 1.97 ±0.21 4.1 ± 0.4 R155I 11.7 ± 2.5  24.0 ± 5.2  R155M 2.68 ± 0.21 5.5 ± 0.4R155G 3.58 ± 0.24 7.4 ± 0.5The stable wild-type (mADE) and variant HCV sub-genomic replicon celllines were generated using the T7 RNA runoff transcripts from thecorresponding high efficiency Con1 replicon plasmids. The average IC₅₀values and SD of telaprevir were determined for the HCV replicon celllines in the 48-h assay in three independent experiments. Fold changewas determined by dividing the IC₅₀ of a given variant by that of thewild-type HCV replicon.

2) Substitutions at Arg¹⁵⁵ of the HCV NS3 Protease Resulted in aDecreased Sensitivity to Telaprevir in Enzyme Assays

To confirm whether substitutions at Arg¹⁵⁵ in the HCV NS3 proteasedomain are sufficient to cause a loss of sensitivity to telaprevir atthe enzyme level, Arg¹⁵⁵ was replaced with Lys, Thr, Ser, or Ile in anNS3 protease domain (genotype 1a) from which the sequences were derivedfrom HCV samples in a patient prior to dosing with telaprevir. The NS3serine protease domain proteins containing R155K, R155T, R155S, or R155Imutations were expressed in E. coli and purified prior to determinationof enzyme sensitivity to telaprevir. Resistance to telaprevir wasdefined by the fold-change in K_(i(app,1h)), which is the apparent K_(i)measured after a 1-h pre-incubation with telaprevir.

A comparison of the sensitivity of telaprevir for the wild-type HCVprotease domain co-complexed with the KK4A cofactor peptide versusvariants with substitutions at Arg¹⁵⁵ is shown in Table II. The averageK_(i(app,1h)) value of telaprevir against the wild-type genotype 1apatient NS3 protease domain complexed with the KK4A peptide was0.044±0.033 μM (Table II). In contrast, the average K_(i(app,1h)) valuesof telaprevir were about 11-fold higher for the R155K protease and9-fold higher for the R155T protease (Table 11), the two major variantsobserved in the phase 1b trial of telaprevir alone. R155S and R155I, twoof the minor Arg¹⁵⁵ variants observed in the telaprevir phase 1b trial,showed 22-fold and 16-fold increases in K_(i(app,1h)) values,respectively, compared to that of wild-type protease (Table II). Thesedata indicate that substitutions of Arg¹⁵⁵ with different amino acids,including a conservative substitution of another positively chargedresidue (Lys), results in a decreased sensitivity to telaprevir.

TABLE II Demonstration of Resistance in HCV NS3 Protease Enzyme AssaysVariants K_(i(app, 1 h)) of telaprevir (μM) Fold change Wild-type (n =5) 0.044 ± 0.033 1.0 ± 0.8 R155K (n = 4) 0.49 ± 0.22 11 ± 5  R155T (n =5) 0.38 ± 0.18 8.7 ± 4.2 R155S (n = 3) 0.97 ± 0.70 22 ± 16 R155I (n = 3)0.71 ± 0.35 16 ± 8 The average K_(i(app,1h)) values and SD of telaprevir were determinedfor the purified wild-type and for four variant HCV NS3 serine proteasedomains using the KK4A cofactor peptide and the FRET substrate in threeto five independent experiments. Fold change was determined by dividingthe K_(i(app,1h)) of a given variant by that of the wild-type protease.

3) X-ray Structure of the R155K HCV NS3 Protease

To understand why substitution of Arg¹⁵⁵ with another residue, includinga positively charged amino acid (Lys), results in a loss of sensitivityto telaprevir, the X-ray crystal structure of the R155K NS3 protease wasdetermined. The R155K mutation was engineered into a T7-tag fused HCV-Hprotease construct that had previously been used to determinate thestructure of the wild-type protease co-complexed with NS4A cofactorpeptide. The ensemble structure of the R155K protease domainco-complexed with NS4A cofactor peptide, with a resolution of 2.5 Å, isvery similar to that of wild-type HCV-H strain protease complexdescribed previously (Kim et al., supra). Briefly, one molecule of NS3protease domain (residues 1 to 181) and one molecule of the co-factorNS4A (residues 21 to 39) form the globular entity, which, in turn, formsthe homodimer with another globular entity in the asymmetrical unit. Oneglobular unit of the Lys¹⁵⁵ variant protease in complex with the NS4Acofactor was superimposed with the wild-type Arg¹⁵⁵ co-complex. Becausethe rms deviation of Cα atoms was only 0.314 Å, there is littledifference in structure of these two proteases.

A close-up view of the side chains of NS3 protease residues 123, 168 and155 in the S2 and S4 pockets is shown in FIG. 25(B). The overall shiftof residue 155 side-chain (Lys¹⁵⁵ versus Arg¹⁵⁵) is small as evidencedby the distance between the Cδ of residue 155 and the Cβ of the Asp⁸¹,one of the catalytic triad residues: 4.26 Å for Lys¹⁵⁵ and 4.24 Å forArg¹⁵⁵. In the R155K protease, the distance between the terminal amine(Nz) of Lys¹⁵⁵ and the Cβ of Asp⁸¹ is 3.5 Å, and the distance betweenthe Cδ of Lys¹⁵⁵ and the Cβ of Asp⁸¹ is 3.6 Å. In the wild-typeprotease, the NH1 and Nz of Arg¹⁵⁵ are 5.6 Å and 5.3 Å, respectively,away from the Cβ of its Asp⁸¹. Thus, the terminal amine group of Lys¹⁵⁵in the R155K protease is closer to the carboxyl group of Asp⁸¹ than thecomparative terminal azide group of Arg¹⁵⁵. In contrast, the distancebetween Nz of Lys¹⁵⁵ and the carboxyl atom Oε2 of Asp¹⁶⁸ is 5.8 Å in theR155K protease compared to 3.2 Å between the corresponding pair of theterminal NH2 of Arg¹⁵⁵ and the Oε2 of Asp¹⁶⁸ in the wild-type protease.Thus, the terminal amine group of Lys¹⁵⁵ in the R155K protease isfurther away from the carboxyl group of Asp¹⁶⁸ than the comparativeterminal azide group of Arg¹⁵⁵. Therefore, substitution of Arg with Lysat residue 155 alters the shape of the S2 binding pocket such that thepositive charge at the Nz atom of Lys¹⁵⁵ can not be neutralized by theadjacent side-chain of Asp¹⁶⁸ as is the case of the wild-type Arg¹⁵⁵ andAsp¹⁶⁸ pair.

4) Mechanism of Resistance of R155K or R155T Variant to Telaprevir

A structural model of the interactions between telaprevir and NS3protease has previously been described (e.g., Lin et al., supra). In amodel of the co-complex of telaprevir with the R155K enzyme, the sameinteractions were maintained except for differences at the side-chain ofresidue 155. In the wild-type protease structure, the Arg¹⁵⁵ side-chainbends over the bicyclic P2 group of telaprevir to make several directvan der Waals contacts, and provides a hydrophobic environment for theP2 group of telaprevir (FIG. 26(A)). However, the Lys¹⁵⁵ side-chain ofthe R155K enzyme has an extended conformation and makes only one or twodirect contacts with the P2 group, thus leaving the P2 group oftelaprevir more exposed to the solvent (FIG. 26(B)). This observation isconsistent with the R155K enzyme being less sensitive to telaprevir, asshown with in vitro enzyme assays. It is reasonable to assume that theR155M variant will have an extended conformation of Met¹⁵⁵ side-chain,and therefore similarly fewer interactions with the P2 group oftelaprevir, consistent with observed decrease in binding of theinhibitor.

To understand the mechanism of binding of telaprevir to variants withresidues with shorter side-chains at position 155, the model of R155Tvariant enzyme complexed with telaprevir was used. It is obvious fromthe model that Thr¹⁵⁵ side-chain is too short to provide a hydrophobiccover for the P2 group of the inhibitor (FIG. 26(C)). Other shorterside-chains like Ile, Ser and Gly are similar or even shorter in sizeand are expected to lose interactions with the P2 group and have adecreased binding affinity to the telaprevir.

5) HCV Variant Replicons with Substitutions at Arg¹⁵⁵ of the NS3Protease Remain Fully Sensitive to IFN-α

Whether the telaprevir-resistant variant replicon cells withsubstitution at NS3 residue 155 remain sensitive to IFN-α or ribavirinwas also determined. As shown in Table III, the IC₅₀ of either IFN-α orribavirin remained virtually the same for HCV replicon cells containingR155K, R155T, or R155M mutations compared to the wild-type repliconcells. These results suggest that combination with IFN-α with or withoutribavirin could be a potential therapeutic strategy to suppress theemergence of HCV variants with substitutions at NS3 protease residue155.

TABLE III Lack of Resistance to Other Anti- HCV Agents in HCV RepliconCells Replicon IC₅₀ Variants IFN-α (U/ml) Fold change Ribavirin (μM)Fold change Wild-type 11.6 ± 1.1  1.0 ± 0.1 58 ± 18 1.0 ± 0.3 R155K 15.2± 12.3 1.3 ± 1.1 37 ± 17 0.6 ± 0.3 R155T 4.8 ± 3.3 0.4 ± 0.3 32 ± 18 0.6± 0.3 R155M 4.9 ± 1.0 0.4 ± 0.1 39 ± 5  0.7 ± 0.1The stable wild-type and variant HCV sub-genomic replicon cell lineswere generated using the 17 RNA runoff transcripts from thecorresponding high efficiency Con1 replicon plasmids. The average IC₅₀values and SD of IFN-α and ribavirin were determined for the HCVreplicon cell lines in the 48-h assay in three independent experiments.Fold change was determined by dividing the IC₅₀ of a given variant bythat of the wild-type HCV replicon.

Example 20 Summary of the Study Results

One study was to monitor the possible emergence of drug resistantmutations to VX-950 monotherapy by sequence analysis of the HCV proteaseNS3-4A region in subjects with genotype 1 hepatitis C who were dosedwith VX-950 for 14 days. Traditionally, resistance genotyping has beendone by population-based sequencing, which detects the dominant sequencein the plasma virus. Any sequences that constitute less than 20% of theviral population will not be detected by this method. Becausedrug-resistance mutations may take longer than 14 days to accumulate tothis measurable level, a new method was developed to detect minorpopulations of variants. Sequences were obtained from about 80-85individual viral clones per subject per time point, so that resistantmutations that may emerge in 14 days of dosing with VX-950 with asensitivity of down to about 5% of the population can be identified.

In subjects grouped by viral response to VX-950, distinct mutationalpatterns were observed. In the first group of subjects whose HCV RNArebounded during the dosing period, wild-type virus was almostcompletely replaced by 1 of 3 viral variants containing a mutation atposition 36, 54, or 155 at ETR and follow up. A V36A mutation was foundin genotype 1b subjects, whereas a V36A/M or R155K/T was seen in thegenotype 1a subjects. Some variants also contained a double mutation atpositions 36 and 155 in 1a subjects. A T54A mutation was seen in both 1aand 1b subtypes. The mutations at positions 36 and 54 appear to bemutually exclusive as they were rarely found together in the samegenome. A second group of subjects had an initial HCV RNA decline thatleveled off at the end of the 14-day dosing period. These subjectsharbored virus that contained a mutation at position 156 from an alanineto either a valine (A156V) or threonine (A156T). This mutation atposition 156 has previously been shown to develop in vitro in thepresence of VX-950 (6, 7). Some subjects harbored a few variants thatalso contained a double mutation at positions 36 and 156.

Subject-specific protease clones were expressed and tested forinhibition by VX-950. There were no significant differences in theenzyme IC₅₀ values of the baseline proteases derived from differentsubject isolates within a subtype. However, the IC50 values of themutant proteases indicate varying degrees of decreased sensitivity toVX-950. HCV RNA in the last group of subjects continued to declinethroughout the dosing period, and some reached levels below the limit ofdetection of the current assay (10 IU/mL). Due to the limit ofsensitivity of our sequencing assay (>100 IU/mL), no viral sequence dataare available for these subjects at Day 14 of dosing. However, samplestaken 7 to 10 days after the last dose of VX-950 were successfullysequenced for all subjects. In the follow-up samples from the first tworesponse groups, resistant mutations were found to persist in the plasmaof all subjects. However, in many cases, the frequency of mutation atposition 156 was significantly decreased, as the wild-type as well asthe mutations at position 36 or 54 began to increase in proportion. Theproportion of virions with a mutation at position 155 remainedrelatively constant. These shifts in mutation patterns are likely due tofitness differences between variants in the absence of drug selectivepressure. It appears that viruses with mutation at positions 36, 54, or155, although less fit than wild-type, are still reasonably fit, whereasthe residue 156 mutants are quite unfit in the absence of drug. Fromthese data, there seems to be an inverse correlation between the levelof resistance and the fitness for different single mutants. The dataderived from the group of subjects with a plateau in plasma HCV RNAindicated the influences of virologic resistance and fitness inproducing a given clinical response. Thus, the clinical response cannot,itself, indicate the underlying virology.

Analysis of the last group of subjects who had continued HCV RNA declineduring dosing and reached low levels of HCV RNA reveals that viruspresent at follow-up consisted of mostly low level resistance mutationsat positions 36 and 54, and 3 of the subjects harbored few mutations butwere mostly or completely wild-type. Although it is unknown whatvariants, if any, were present at Day 14, this result suggests that withan optimal response to VX-950, it may be possible to avoid clinicalresistance with monotherapy or by the addition of other antiviralcompounds such as Peg-IFN. Optimizing dosing regimens may extend thisresponse to a greater number of patients.

In summary, these results indicate that the dosing regimen of VX-950 inthis study can result in the selection of different mutations in the NS3protease with varying levels of drug resistance. Increasedconcentrations of VX-950 are expected to prevent emergence of virus withlow level resistance (at positions 36, 54, and 155). The remaining highlevel resistant virus (at position 156) may be overcome throughdifferent treatment options, including combination therapy. Higher drugconcentrations may inhibit viral replication more completely, causing asteeper slope of initial decline; thus reducing the chance thatresistant mutations will be selected and cause clinical resistance. Theaddition of Peg-IFN to VX-950 treatment may enhance immune-mediatedclearance of the virus, and the effectiveness of immune-mediatedclearance should not be affected by the presence of resistant variants.Although the mutation at position 156 confers high levels of resistance,it appears to come with significant fitness costs, as measured by itsrelative rate of replication in the absence of VX-950. There isincreasing evidence that antiviral drug resistance is associated withimpaired viral fitness, which can translate into a clinical benefit(1-3, 9). Resistant virus replicating at such a low level may notaccumulate compensatory fitness mutations immediately, allowing the hostimmune system or other drugs, such as Peg-IFN, to clear the remainingvirus.

REFERENCES

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TABLE 1 Mutations in the HCV Protease Alone or in Combination FoundAfter Dosing with VX-950 Subject Subj VX-950 Geno- Δ in Time %Single^(c) % Double^(c) Average Group ID Dose type VL^(a) Point^(b) V36T54 R155 A156 36/155 36/156 Phenotype^(d) Rebound 2101 450 1a 2.56ETR^(e) 9 4 13 0 77 0 36.70 FU^(f) 23.5 0 11.5 0 60.5 0 29.43 2102 4501a 1.84 ETR 35 3 32 8 12 3 69.69 FU 31 0 26 0 26 0 14.81 2105 450 1b1.06 ETR 86 0 0 5 0 0 26.31 FU 45 32 0 6 0 0 28.94 2107 450 1b 2.09 ETR n/a^(g) n/a n/a n/a n/a n/a n/a FU n/a n/a n/a n/a n/a n/a n/a 3108 4501b 2.08 ETR 66 31 0 0 0 0 6.03 FU 62 8 0 0 0 0 3.13 3111 450 1b 1.82 ETR24 67 0 2 0 1 30.71 FU 31 63 0 0 0 0 8.65 2211 750 1a 2.35 ETR n/a n/an/a n/a n/a n/a n/a FU 35 1 31 0 13 0 9.33 2310 1250 1a 0.86 ETR 42 0 330 14 0 10.15 FU 41 1 22 0 14 0 9.39 2312 1250 1a 3.20 ETR 38 14 9 0 52 026.13 FU 36 9 10 0 52 0 26.03 3301 1250 1a 1.16 ETR 8 4 65 0 10 0 9.38FU 28 0 34 0 16 0 10.65 3302 1250 1a 2.50 ETR 39 8 15 0 24 0 14.39 FU 3110 26 0 18 0 12.33 3303 1250 1a 1.93 ETR 0 1 79 0 20 0 14.59 FU 6 0 66 023 0 15.28 3305 1250 1a 2.25 ETR 63 2 10 0 12 4.5 47.49 FU 77 0 4 0 10 115.38 Plateau 2104 450 1a 0.65 ETR 34 0 27 4 9 4 57.05 FU 82 0 0 0 8 06.55 2106 450 1b 0.41 ETR n/a n/a n/a n/a n/a n/a n/a FU 5 8 0 0 0 01.14 3201 750 1a 0.73 ETR 0 0 2 100 0 0 466.14 FU 25 2 24 3 22 3 45.743202 750 1b 0.22 ETR 0 0 0 100 0 0 466.00 FU 24 16 0 29 0 2 126.48 123081250 1b 0.05 ETR 0 68 0 30 0 0 147.96 FU 7 64 0 0 0 0 7.93 2309 1250 1b0.33 ETR n/a n/a n/a n/a n/a n/a n/a FU 19 10 0 0 0 0 1.87 2311 1250 1a0.62 ETR 72 92 12 2 8 1 25.96 FU n/a n/a n/a n/a n/a n/a n/a 3306 12501b 0.26 ETR 0 0 0 100 0 0 466.00 FU 52 25 0 4 0 1 30.97 Continued 3110450 1b — ETR  nd^(h) nd nd nd nd nd nd Response FU 21 53.5 0 0 0 0 6.803112 450 1b — ETR nd nd nd nd nd nd nd FU 3 6 0 0 0 0 0.83 2207 750 1b —ETR nd nd nd nd nd nd nd FU n/a n/a n/a n/a n/a n/a n/a 3203 750 1b —ETR nd nd nd nd nd nd nd FU 9 1 0 2 0 0 9.65 3204 750 1b — ETR nd nd ndnd nd nd nd FU 25 20 0 0 0 0 3.17 3205 750 1a — ETR nd nd nd nd nd nd ndFU 62 11 21 0 5 0 4.92 3212 750 1b — ETR nd nd nd nd nd nd nd FU 0 0 0 00 0 0.00 ^(a)Log change in HCV RNA from nadir to Day 14 (end of dosing).Patient 3108 did not have a Day 14 HCV RNA value, and so it was inferredusing Day 11 and Day 17 values. ^(b)Days after first dose of VX-950^(c)Percentages base on an average of 82 clones ^(d)Sum of (% mutant atamino acid position) × (average fold change in IC₅₀ for amino acidposition for all single and double mutants/100 ^(e)ETR = end oftreatment (Day 14) ^(f)FU = follow up (7-10 days after ETR) ^(g)n/a =not available (in progress) ^(h)nd = not detectable

TABLE 2 IC₅₀ Analysis of Single and Double Mutants with VX-950 and BILN2061 VX950 BILN2061 IC₅₀ Fold IC₅₀ Fold Mutation [nM] change [nM] changeV36M 110 1.7  nd* nd V36M 280 4.4 2.3 0.5 V36M 156 2.4 2.0 0.4 V36L 1402.2 2.1 0.5 V36A 125 2.0 nd nd V36A 275 4.3 2.3 0.5 V36A 250 3.9 26 5.5V36A 264 4.1 nd nd V36A 444 6.9 9.1 2.0 T54S 120 1.9 3.6 0.8 T54A 749 1210 2.3 R155K 275 4.3 632 137 R155K 300 4.7 nd nd R155K 410 6.4 >840 >183R155M 425 6.6 nd nd R155S 370 5.8 >840 >183 R155T 335 5.2 nd nd R155T465 7.3 696 151 R155T 915 14 799 174 A156S 1400 22 7.4 1.6 A156T 21500336 >840 >183 A156T 15000 234 nd nd A156I >50000 >781 nd ndA156V >50000 >781 nd nd A156V 12500 195 nd Nd V36A, R155K 1350 21 nd ndV36A, R155K 1800 28 nd nd V36M, R155K 3593 56 nd nd V36M, R155K 295046 >840 >183 V36M, R155K 4043 63 nd nd V36M, R155T 3810 60 729 158 V36M,A156T >50000 >781 nd nd *nd = not determined

TABLE 3 Mean IC₅₀ values for VX-950 of Different Amino Acid Mutations atthe Same Position IC₅₀ IC₅₀ Fold Fold mean SD change change Mutation[nM] [nM] mean SD V36M/L/A 227 106 3.5 1.7 T54S 120 1.9 T54A 749 12R155K/M/S/T 437 204 6.8 3.2 A156S 1400 22 A156T/V/I 29800 18730 466 293V36M/A, R155K/T 2924 1116 46 17 V36M, A156T >50000 >781

TABLE 4 Fitness of Viral Mutants Fold Reduction in Fitness FitnessRelative to (wild-type Viral Mutation Fitness Wild-type set to 100) None(wild-type) 4.17 — 100 V36A/M 2.82 −1.48 68 R155K/T + V36A/M 2.0 −2.0948 T54A 1.86 −2.24 45 R155K/T 1.58 −2.64 38 A156V/T 0.1 −41.70 2.4

TABLE 5 Summary of Resistance of Mutant HCV Proteases VX-950 SCH 503034BILN 2061 ITMN-191 Viral Replicon Enzyme Replicon Enzyme Replicon EnzymeReplicon Enzyme Variant Assay^(a) Assay^(b) Assay Assay Assay AssayAssay Assay V36M 7.0 (1.6) 5.8 2.7 (0.9) 4.4 1.4 (0.9) 0.6 2.1   1.9V36A 7.4 (2.2) — 3.2 (0.9) — 1.7 (1.1) — 1.5 — V36G 11.2 (0.4) — 2.4(0.3) — 1.1 — 1.7 — V36L 2.2 (0.4) 2.2 1.1 (0.3) — 1.5 — 0.9 — T54A 6.3(1.7) — 3.2 (1.1) — 0.9 (0.2) — 1.0 — V36A + T54A 20.1 (2.9) — 4.5 (0.2)— 0.6 (0.3) — 0.5 (0.1) — R155K 7.4 (0.6) 11 6.2 (4.9) 10 355 (213) >30062.8  120  R155T 19.8 (1.8) 8.7 10.2 (2.4) — 645 (173) 72 9.2 — R155S4.1 (0.4) 22 1.8 (0.6) — 592 (124) >310 8.0 (0.2) — R155I 24.0 (5.2) 166.6 (2.1) — 36.5 (9.5) 56 1.3 (0.2) — R155M 5.5 (0.4) — 2.5 (0.3) — 42.2(2.1) — — — R155G 7.4 (0.5) — 2.8 (0.6) — 821 (208) — 18.7 (3.0) —V36A + R155K ~40 — 8.3 (0.7) — 757 (99) — 316 (191) — V36A + R155T >62 —21.9 (6.6) — 835 (91) — 33.0  — V36M + R155K ~63 74 11.3 (5.5) 39 791(343) >250 263 (155) 220  V36M + R155T >62 — 21.3 (2.6) — 1160 (110) —66.7  — V36M + A156T >62 — >37 — 1986 (1104) — 12.0 (2.0) — A156S — 50 —37 — 9.7 10 A156T — 410 — 310 — >310 12 V170A 2.6 (0.6) 3.3 (0.6) 1.8(0.3) ?? R109K 0.8 (0.2) 0.8 (0.2) 1.2 (0.4) ??

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject disclosure have beendiscussed, the above specification is illustrative and not restrictive.Many variations of the disclosure will become apparent to those skilledin the art upon review of this specification and the claims below. Thefull scope of the disclosure should be determined by reference to theclaims, along with their full scope of equivalents, and thespecification, along with such variations

1-51. (canceled)
 52. An isolated Hepatitis C Virus (HCV) NS3 protease,wherein said protease is proteolytically active, wherein said proteaseis encoded by a polynucleotide having a nucleic acid sequence that is atleast 60% identical to the nucleic acid sequence of SEQ ID NO: 1 fromnucleotide position 1 through nucleotide position 543, wherein saidnucleic acid sequence encodes an amino acid sequence having at least oneamino acid alteration, said amino acid alteration being at the aminoacid position corresponding to amino acid position 54 of SEQ ID NO: 2.53. The HCV protease of claim 52, wherein the amino acid alterationresults in a serine at the position corresponding to amino acid position54 of SEQ ID NO:
 2. 54. The isolated HCV protease of claim 52, whereinthe protease has at least one additional amino acid alteration at anamino acid position selected from the group consisting of amino acidpositions corresponding to amino acid positions 36, 155 and 156 of SEQID NO: 2, and combinations thereof.
 55. An isolated Hepatitis C Virus(HCV) NS3 protease, wherein said protease is proteolytically active,wherein said protease is encoded by a polynucleotide having a nucleicacid sequence that is at least 60% identical to the nucleic acidsequence of SEQ ID NO: 1 from nucleotide position 1 through nucleotideposition 543, wherein said nucleic acid sequence encodes an amino acidsequence having at least one amino acid alteration, said amino acidalteration being at the amino acid position corresponding to amino acidposition 36 of SEQ ID NO: 2, wherein said amino acid alteration resultsin either a methionine or glycine at the amino acid positioncorresponding to position 36 of the HCV NS3 protease amino acid sequenceof SEQ ID NO:
 2. 56. The isolated HCV protease of claim 55, wherein theprotease has at least one additional amino acid alteration at an aminoacid position selected from the group consisting of amino acid positionscorresponding to amino acid positions 54, 155 and 156 of SEQ ID NO: 2,and combinations thereof.
 57. An isolated Hepatitis C Virus (HCV) NS3protease, wherein said protease is proteolytically active, wherein saidprotease is encoded by a polynucleotide having a nucleic acid sequencethat is at least 60% identical to the nucleic acid sequence of SEQ IDNO: 1 from nucleotide position 1 through nucleotide position 543,wherein said nucleic acid sequence encodes an amino acid sequence havinghas at least one amino acid alteration, said amino acid alteration beingat the amino acid position corresponding to amino acid position 155 ofSEQ ID NO: 2, said amino acid alteration resulting in an amino acidselected from the group consisting of lysine, threonine, serine,isoleucine, methionine, leucine and glycine at the amino acid positioncorresponding to position 155 of the HCV NS3 protease amino acidsequence of SEQ ID NO:
 2. 58. The isolated HCV protease of claim 57,wherein the protease has at least one additional amino acid alterationat an amino acid position selected from the group consisting of aminoacid positions corresponding to amino acid positions 36, 54 and 156 ofSEQ ID NO: 2, and combinations thereof.
 59. An isolated Hepatitis CVirus (HCV) NS3 protease, wherein said protease is proteolyticallyactive, wherein said protease is encoded by a polynucleotide having anucleic acid sequence that is at least 60% identical to the nucleic acidsequence of SEQ ID NO: 1 from nucleotide position 1 through nucleotideposition 543, wherein said nucleic acid sequence encodes an amino acidsequence having at least one amino acid alteration, said amino acidalteration being at the amino acid position corresponding to amino acidposition 156 of SEQ ID NO: 2, said amino acid alteration resulting inisoleucine at the amino acid position corresponding to position 156 ofthe HCV NS3 protease amino acid sequence of SEQ ID NO:
 2. 60. Theisolated HCV protease of claim 59, wherein the protease sequence has atleast one additional amino acid alteration at an amino acid positionselected from the group consisting of amino acid positions correspondingto amino acid positions 36, 54 and 155 of SEQ ID NO: 2, and combinationsthereof.
 61. An isolated Hepatitis C Virus (HCV) NS3 protease, whereinsaid protease is capable of binding to either a VX-950 proteaseinhibitor or a BILN 2061 protease inhibitor in the presence of HCV NS4Apeptide cofactor, and wherein said protease is encoded by apolynucleotide having a nucleic acid sequence that is at least 60%identical to the nucleic acid sequence of SEQ ID NO: 1 from nucleotideposition 1 through nucleotide position 543, wherein said nucleic acidsequence encodes an amino acid sequence having at least one amino acidalteration at an amino acid position selected from the group of aminoacid positions corresponding to amino acid positions 36, 54, 155 and 156of SEQ ID NO: 2, (i) wherein said amino acid alteration at the aminoacid position corresponding to amino acid position 36 of SEQ ID NO: 2,if it is the only amino acid alteration at a position corresponding toone of the amino acid positions 36, 54, 155 and 156, results in amethionine or glycine, (ii) wherein said amino acid alteration at theamino acid position corresponding to amino acid position 54 of SEQ IDNO: 2, if it is the only amino acid alteration at a positioncorresponding to one of the amino acid positions 36, 54, 155 and 156,results in a serine, (iii) wherein said amino acid alteration at theposition corresponding to amino acid position 155 of SEQ ID NO: 2, if itis the only amino acid alteration at a position corresponding to one ofthe amino acid positions 36, 54, 155 and 156, results in an amino acidselected from the group consisting of lysine, threonine, serine,isoleucine, methionine, leucine and glycine, and (iv) wherein said aminoacid alteration at the position corresponding to amino acid position 156of SEQ ID NO: 2, if it is the only amino acid alteration at a positioncorresponding to one of the amino acid positions 36, 54, 155 and 156,results in an amino acid encoding isoleucine.
 62. The isolated HCVprotease of claim 61, wherein the protease sequence comprises a firstamino acid alteration, said amino acid alteration being at the aminoacid position corresponding to position 36 of SEQ ID NO: 2, and whereinthe protease further comprises at least an additional amino acidalteration at one or more amino acid positions selected from the groupof amino acid positions corresponding to amino acid positions 54, 155and
 156. 63. The isolated HCV protease of claim 61, wherein the proteasesequence comprises a first amino acid alteration, said amino acidalteration being at the amino acid position corresponding to position 54of SEQ ID NO: 2, and wherein the protease further comprises at least anadditional amino acid alteration at one or more amino acid positionsselected from the group of amino acid positions corresponding to aminoacid positions 36, 155 and
 156. 64. The isolated HCV protease of claim61, wherein the protease sequence comprises a first amino acidalteration, said amino acid alteration being at the amino acid positioncorresponding to position 155 of SEQ ID NO: 2, and wherein the proteasefurther comprises at least an additional amino acid alteration at one ormore amino acid positions selected from the group of amino acidpositions corresponding to amino acid positions 36, 54 and
 156. 65. Theisolated HCV protease of claim 61, wherein the protease sequencecomprises a first amino acid alteration, said amino acid alterationbeing at the amino acid position corresponding to position 156 of SEQ IDNO: 2, and wherein the protease further comprises at least an additionalamino acid alteration at one or more amino acid positions selected fromthe group of amino acid positions corresponding to amino acid positions36, 54 and
 155. 66. The HCV protease of any one of claims 52-60, whereinsaid nucleic acid sequence has at least 90% sequence identity with theregion of the nucleic acid sequence of SEQ ID NO: 1 from nucleotideposition 1 through nucleotide position
 543. 67. The HCV protease ofclaim 66, wherein said nucleic acid sequence has at least 95% sequenceidentity with the region of the nucleic acid sequence of SEQ ID NO: 1from nucleotide position 1 through nucleotide position 543.